TWI445547B - Dengue virus peptide vaccine and methods of preparing and using the same - Google Patents

Dengue virus peptide vaccine and methods of preparing and using the same Download PDF

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TWI445547B
TWI445547B TW097120818A TW97120818A TWI445547B TW I445547 B TWI445547 B TW I445547B TW 097120818 A TW097120818 A TW 097120818A TW 97120818 A TW97120818 A TW 97120818A TW I445547 B TWI445547 B TW I445547B
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Hsin Wei Chen
Chih Hsiang Leng
Pele Choi-Sing Chong
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Description

登革熱病毒胜肽疫苗及其製備與使用方法Dengue virus peptide vaccine and preparation and use method thereof

總體而言，本發明係關於登革熱病毒學，更特定而言，係關於登革熱病毒胜肽疫苗以及製備及使用該疫苗之方法。In general, the present invention relates to dengue virology, and more particularly to dengue virus peptide vaccines and methods of making and using the vaccines.

登革熱(DF)為瘧疾之後影響人類的最主要蚊傳疾病。登革熱為一種自限性發燒疾病，由登革熱病毒(DEN)之四種密切相關的血清型(登革熱-1型、登革熱-2型、登革熱-3型及登革熱-4型)之任一種所引起，其經由蚊子傳給人類。在大多數病例中，罹患登革熱之病人會突然發燒，具有嚴重的頭痛、肌肉及關節疼痛，並且偶而會出現皮疹。登革熱可能會惡化成出血性登革熱(DHF)，DHF可能呈現高燒、出血現象、血小板減少及血液濃縮。小部分的病例將導致登革熱休克症候群(DSS)，其具有高死亡率。Dengue fever (DF) is the most important mosquito-borne disease affecting humans after malaria. Dengue fever is a self-limiting fever caused by any of four closely related serotypes of dengue virus (DEN) (dengue type-1, dengue type-2, dengue type-3, and dengue type-4). It is transmitted to humans via mosquitoes. In most cases, patients with dengue fever have a sudden fever, severe headache, muscle and joint pain, and occasionally a rash. Dengue fever may worsen into hemorrhagic dengue fever (DHF), which may present with high fever, bleeding, thrombocytopenia, and blood concentration. A small number of cases will result in dengue shock syndrome (DSS), which has a high mortality rate.

登革熱感染的臨床診斷通常可於臨床進行。若臨床上顯示罹患登革熱，則血清學及聚合酶連鎖反應(PCR)研究可用來確認該登革熱之診斷。具有較輕微症狀之一些病例，例如未出現皮疹時，可能被誤診為感冒或其他病毒感染。登革熱感染之正確診斷就適當治療該病而言頗為重要。例如，雖然阿斯匹靈及非類固醇消炎藥劑常用於治療病毒感染所引起之疼痛及發燒，但其會加重與一些登革熱感染相關之出血傾向。應使用乙醯胺酚(acetaminophen)替代阿斯匹靈及非類固醇消炎藥劑來治療此等症狀。The clinical diagnosis of dengue infection is usually performed clinically. Serological and polymerase chain reaction (PCR) studies can be used to confirm the diagnosis of dengue if clinically indicated to have dengue fever. Some cases with milder symptoms, such as no rash, may be misdiagnosed as a cold or other viral infection. The correct diagnosis of dengue infection is important in the proper treatment of the disease. For example, although aspirin and non-steroidal anti-inflammatory agents are commonly used to treat pain and fever caused by viral infections, they may aggravate the bleeding tendency associated with some dengue infections. Acetaminophen should be used in place of aspirin and non-steroidal anti-inflammatory agents to treat these symptoms.

證據顯示出血性登革熱(DHF)比較可能在由血清型與原發性感染不同之繼發性感染之病患中發生，該現象亦稱為重複感染(superinfection)。此係由於抗體，依存性加強作用(ADE)，該加強作用使得在被不同病毒株繼發性感染期間， 攝入作用(uptake)及病毒粒子複製增加。所以，較佳情況係任何有潛力的登革熱疫苗對多種血清型有效，最佳為對所有四種血清型有效。對所有四種血清型無效之登革熱疫苗會使病患個人面臨發生DHF及/或DSS之風險。Evidence suggests that hemorrhagic dengue fever (DHF) is more likely to occur in patients with secondary infections that differ from serotypes to primary infections, a phenomenon known as superinfection. This is due to the antibody-dependent potentiation (ADE), which enhances the secondary infection of different strains of virus, Uptake and increased virion replication. Therefore, it is preferred that any potential dengue vaccine is effective against multiple serotypes, preferably for all four serotypes. A dengue vaccine that is ineffective against all four serotypes exposes the individual to the risk of developing DHF and/or DSS.

支持性療法為DF之主要治療。利用經口攝取之流體，補充以靜脈輸注流體，來預防脫水及顯著的血液濃縮。Supportive therapy is the main treatment for DF. The fluid ingested orally is supplemented with an intravenous infusion fluid to prevent dehydration and significant blood concentration.

目前，登革熱病毒感染之初步預防措施主要為清除或減少登革熱之媒介物-蚊子。期望開發對登革熱病毒之多種血清型有效之疫苗以及登革熱感染之簡單且更正確的診斷試驗。本發明之具體實施例係關於該疫苗及診斷試驗。At present, the initial preventive measures for dengue virus infection are mainly to remove or reduce the mediator of dengue fever - mosquitoes. It is desirable to develop vaccines that are effective against multiple serotypes of dengue virus, as well as simple and more accurate diagnostic tests for dengue infection. Specific embodiments of the invention relate to the vaccine and diagnostic assays.

根據本發明，已設計及製造出胜肽疫苗，以誘生中和性抗體反應，對抗多種血清型登革熱病毒。In accordance with the present invention, a peptide vaccine has been designed and manufactured to induce a neutralizing antibody response against a variety of serotype dengue viruses.

一廣義方面，本發明之具體實施例係關於一種主要係由序列識別號：5所組成之單離、重組或合成胜肽。In a broad aspect, a particular embodiment of the invention pertains to a single, recombinant or synthetic peptide consisting essentially of sequence identifier:5.

另一方面，本發明之具體實施例係關於一種免疫組成物。該免疫組成物包括主要係由序列識別號：5組成之胜肽。在一具體實施例中，該免疫組成物包括主要係由序列識別號：5組成之胜肽及佐劑。本發明之另一具體實施例係關於一種套組，其包括該免疫組成物以及說明使用該免疫組成物以誘發受治者免疫反應之說明書。本發明之另一具體實施例係關於誘發受治者免疫反應之方法。該方法包括對該受治者投予具免疫有效量之免疫組成物。本發明之再一具體實施例係關於一種預防受治者產生與登革熱病毒相關之疾病或異常之的方法。該方法包含對該受治者投予具疫苗接種有效量之疫苗，其中該疫苗包括主要係由序列識別號：5組成之胜肽。In another aspect, particular embodiments of the invention are directed to an immunological composition. The immunological composition includes a peptide consisting essentially of SEQ ID NO: 5. In a specific embodiment, the immunological composition comprises a peptide consisting essentially of SEQ ID NO: 5 and an adjuvant. Another embodiment of the invention is directed to a kit comprising the immunological composition and instructions for using the immunological composition to elicit an immune response in the subject. Another embodiment of the invention is directed to a method of inducing an immune response in a subject. The method comprises administering to the subject an immunologically effective amount of an immunological composition. Still another embodiment of the present invention relates to a method of preventing a subject from developing a disease or abnormality associated with dengue virus. The method comprises administering to the subject a vaccination effective amount of a vaccine, wherein the vaccine comprises a peptide consisting essentially of SEQ ID NO: 5.

另一方面，本發明之具體實施例係關於一種能選擇性與主要係由序列識別號：5所組成之胜肽結合的抗體。本發明之另一具體實施例係關於一種製造能選擇性與主要係由序列識別號：5組成之胜肽結合之抗體的方法。該方法包括使細胞與該主要係由序列識別號：5組成之胜肽接觸；以及於允許該細胞產生該抗體之條件下培養該細胞。本發明之另一具體實施例係關於一種治療組成物，其包括：與主要係由序列識別號：5組成之胜肽選擇性結合的抗體。本發明之另一具體實施例係關於一種治療受治者之登革熱疾病或異常之方法。該方法包括對該受治者投予具治療有效量之治療組成 物，該治療組成物包括與主要係由序列識別號：5組成之胜肽選擇性結合的抗體。In another aspect, a specific embodiment of the invention pertains to an antibody that selectively binds to a peptide consisting essentially of sequence identifier:5. Another embodiment of the invention is directed to a method of making an antibody that selectively binds to a peptide consisting essentially of SEQ ID NO: 5. The method comprises contacting a cell with a peptide consisting of the major sequence: SEQ ID NO: 5; and culturing the cell under conditions that allow the cell to produce the antibody. Another embodiment of the invention is directed to a therapeutic composition comprising: an antibody that selectively binds to a peptide consisting essentially of SEQ ID NO: 5. Another embodiment of the invention is directed to a method of treating a dengue disease or disorder in a subject. The method comprises administering to the subject a therapeutically effective amount of a therapeutic composition The therapeutic composition comprises an antibody that selectively binds to a peptide consisting essentially of SEQ ID NO: 5.

再一方面，本發明之具體實施例係關於檢測受治者之登革熱病毒感染之方法。在一具體實施例中，該方法包括：(a)從該受治者取得到生物檢體；(b)使該生物檢體與主要係由序列識別號：5組成之胜肽選擇性結合的抗體接觸；以及(c)檢測在該生物檢體中與該抗體專一性結合之抗原的存在。在另一具體實施例中，該診斷方法包括：(a)從該受治者取得生物檢體；(b)使該生物檢體與主要係由序列識別號：5組成之胜肽接觸；以及(c)檢測在該生物檢體中與該胜肽專一性結合之抗體的存在。In a further aspect, a specific embodiment of the invention relates to a method of detecting a dengue virus infection in a subject. In a specific embodiment, the method comprises: (a) obtaining a biosample from the subject; (b) selectively binding the biosample to a peptide consisting of a sequence identifier of: Contacting the antibody; and (c) detecting the presence of an antigen that specifically binds to the antibody in the biological sample. In another specific embodiment, the diagnostic method comprises: (a) obtaining a biological sample from the subject; (b) contacting the biological sample with a peptide consisting of a major sequence number: 5; (c) detecting the presence of an antibody that specifically binds to the peptide in the biological sample.

本發明之具體實施例亦關於檢測受治者之登革熱病毒感染之套組。在一實施例中，該套組包括主要係由序列識別號：5組成之胜肽以及說明使用該胜肽檢測在該受治者中登革熱病毒感染的說明書。在另一實施例中，該套組包括選擇性地與主要係由序列識別號：5組成之胜肽結合的抗體以及說明使用該抗體檢測在該受治者中登革熱感染之說明書。Specific embodiments of the invention are also directed to a kit for detecting a dengue virus infection in a subject. In one embodiment, the kit includes a peptide consisting essentially of sequence identifier: 5 and instructions for detecting dengue virus infection in the subject using the peptide. In another embodiment, the kit comprises an antibody that selectively binds to a peptide consisting essentially of SEQ ID NO: 5 and instructions for using the antibody to detect dengue infection in the subject.

本發明之另一方面係關於一種設計對抗登革熱病毒之胜肽疫苗的方法，其中該登革熱病毒係選自第1、2、3及4血清型組成之群中之至少一種登革熱病毒。該方法包括：(a)對於登革熱病毒之包膜蛋白第三區塊之胺基酸序列，決定共有序列；以及(b)設計包括該共有序列之胜肽的胜肽疫苗。Another aspect of the invention relates to a method of designing a peptide vaccine against dengue virus, wherein the dengue virus is selected from at least one of the group consisting of 1, 2, 3 and 4 serotypes. The method comprises: (a) determining a consensus sequence for the amino acid sequence of the third block of the envelope protein of the dengue virus; and (b) designing a peptide vaccine comprising the peptide of the consensus sequence.

本發明之其他方面、特徵及優點從下述揭示內容將顯而易知，該揭示內容包括發明之詳細說明及其較佳具體實施例以及隨附之申請專利範圍。The other aspects, features, and advantages of the present invention will be apparent from the description of the appended claims.

在發明背景及全篇說明書中引用或述及各種刊物、文章及專利，此等文獻之每篇皆以其全文以參考文獻之方式納入本文。本說明書所包括之文件、法案、物質、裝置、物品等之討論皆係為了提供本發明之內容。此等討論非承認此等事項之全部或任一者組成本文所揭示之任何發明或請求專利範圍之先前技術之一部分。Various publications, articles, and patents are cited or recited in the <Desc/Clms Page number> The discussion of documents, acts, substances, devices, articles, and the like, which are included in the specification, are intended to provide the invention. These discussions do not admit that all or any of these matters constitute part of the prior art of any invention or claimed scope disclosed herein.

定義definition

除非另有界定，本文所用之所有技術及科學術語具有本發明相關技術領域具有普通技術人士所通常了解之相同意義。在本案中，某些術語經常使用，此等術語應具有本說明書所規定之意義。必須注意單數形「一」及「該」，當用於本文及隨附之申請專利範圍中時，除非內文清楚指定，否則包括複數的指示對象。Unless defined otherwise, all technical and scientific terms used herein have the same meaning meaning In the present case, certain terms are often used and such terms shall have the meanings specified in this specification. It must be noted that the singular forms "a" and "the" are used in the context of the application and the accompanying claims, unless the context clearly dictates otherwise.

術語「佐劑」用於本文時係指加至免疫組成物諸如疫苗中，同時本身不具有任何單一性抗原作用之物質，其能刺激免疫系統及增加對於免疫組成物之免疫反應。佐劑之例子包括，但不限於，明礬沉澱劑、弗氏完全佐劑(Freund's complete adjuvant)、弗氏不完全佐劑、單磷醯基-脂質A/海藻糖二棒桿黴菌酸酯(trehalose dicorynomycolate)佐劑、含有短小棒狀桿菌(Corynebacterium parvum)及tRNA之水/油型乳液、以及藉由模擬經演化保留分子之特定組合(specific sets)而達成增加免疫反應之工作之其他物質，該等其他物質包括微脂體、脂多醣(LPS)、抗原之分子籠形物、細菌細胞壁之成分以及被胞吞之核酸(諸如雙股RNA、單股DNA及含有未經甲基化CpG二核苷酸之DNA)。The term "adjuvant" as used herein, refers to a substance that is added to an immunological composition, such as a vaccine, while not having any single antigenic effect, which stimulates the immune system and increases the immune response to the immune composition. Examples of adjuvants include, but are not limited to, alum precipitant, Freund's complete adjuvant, Freund's incomplete adjuvant, monophosphonium-lipid A/trehalose trehalose A dicorynomycolate adjuvant, a water/oil emulsion containing Corynebacterium parvum and tRNA, and other substances that work to increase the immune response by mimicking specific sets of evolved retention molecules. Other substances include liposomes, lipopolysaccharide (LPS), molecular clathrates of antigens, components of bacterial cell walls, and endocytosed nucleic acids (such as double-stranded RNA, single-stranded DNA, and unmethylated CpG dinuclears). DNA of glycosidic acid).

術語「抗體」用於本文時係指具有特定胺基酸序列且只與密切相關的抗原或一群抗原結合之免疫球蛋白分子或免疫球蛋白分子之至少一個免疫活性部分。「抗體」之例子包括IgG、IgM、IgA、IgD及IgE。免疫球蛋白分子之免疫活性部分之例子包括Fab及F(ab)'2 片段，其可藉由用酶諸如胃蛋白酶處理抗體而產生。抗體之例子包括抗蛇毒血清(antivenin或antivenom或antivenene)，一種用於毒蛇咬傷或螫傷之生物產品。「抗體」可為單株抗體或多株抗體。術語「單株抗體」係指僅含有一種抗原結合位點且能與特定表位(epitope)進行免疫反應之一群抗體分子。術語「多株抗體」係指含有超過一種抗原結合位點且能與多胜肽上超過1個的表位進行免疫反應之一群抗體分子。The term "antibody" as used herein, refers to an immunoglobulin molecule or immunoglobulin molecule having at least one immunologically active portion that has a particular amino acid sequence and that binds only to a closely related antigen or group of antigens. Examples of "antibody" include IgG, IgM, IgA, IgD, and IgE. Examples of immunologically active portions of immunoglobulin molecules include Fab and F(ab)' 2 fragments which can be produced by treating an antibody with an enzyme such as pepsin. Examples of antibodies include antivenin or antivenom or antivenene, a biological product used for snake bites or bruises. The "antibody" may be a single antibody or a plurality of antibodies. The term "monoclonal antibody" refers to a group of antibody molecules that contain only one antigen binding site and are capable of immunologically reacting with a particular epitope. The term "multi-drug antibody" refers to a group of antibody molecules that contain more than one antigen binding site and are capable of immunologically reacting with more than one epitope on a multi-peptide.

術語「抗原」用於本文時係指含有1個或多個表位之分子，該表位將會刺激宿主之免疫系統而產生體液性及/或細胞性抗原-單一性反應。「抗原」與適當細胞接觸的結果為誘導敏感性(sensitivity)或免疫反應性以及在活體或試管中以可顯現方式與抗體或致敏之受治者之免疫細胞反應。在生物體中，「抗原」可被抗體單一性識別及結合。與主要組織相容性複合物(MHC)締合之抗原亦可被T淋巴球(T-細胞)表面上之受體識別及結合，而導致T-細胞之活化。The term "antigen" as used herein, refers to a molecule containing one or more epitopes that will stimulate the host's immune system to produce a humoral and/or cellular antigen-single reaction. The result of contact of an "antigen" with an appropriate cell is induction sensitivity or immunoreactivity and in a viable form in a living or test tube with the immune cells of the antibody or sensitized subject. In an organism, an "antigen" can be uniquely recognized and bound by an antibody. Antigens associated with major histocompatibility complex (MHC) can also be recognized and bound by receptors on the surface of T lymphocytes (T-cells), resulting in activation of T-cells.

術語「生物檢體」係指得自生物(例如病患)或得自生物之組分(例如細胞)的檢體。該檢體可為任何生物組織、細胞或流體。該檢體可為「臨床檢體」，即衍生自受治者諸如人類病患或動物受治者之檢體。此等檢體包括，但不限於，唾液、痰、血液、血球(例如白血球)、羊水、血漿、***、骨髓、以及組織或細針穿刺活檢(fine needle biopsy)檢體、尿液、腹膜液及胸液、或從該等而來知細胞。生物檢體亦可包括組織切片諸如供組織學應用之冷凍切片。生物檢體亦可稱 作「病患檢體」。「生物檢體」亦可包括經實質純化或單離之蛋白質、膜製劑或細胞培養物。The term "biological sample" refers to a sample obtained from an organism (eg, a patient) or a component derived from a living being (eg, a cell). The specimen can be any biological tissue, cell or fluid. The specimen may be a "clinical specimen", that is, a specimen derived from a subject such as a human patient or an animal subject. Such specimens include, but are not limited to, saliva, sputum, blood, blood cells (eg white blood cells), amniotic fluid, plasma, sperm, bone marrow, and tissue or fine needle biopsy specimens, urine, peritoneal fluid And pleural fluid, or cells from these sources. Biopsies can also include tissue sections such as cryosections for histological applications. Biological specimens can also be called As a "patient's specimen." "Biopsies" may also include substantially purified or isolated proteins, membrane preparations or cell cultures.

術語「共有序列」用於本文時係指藉由將一系列的多個序列對準而決定之胺基酸或核酸序列，該序列界定理想化序列，該理想化序列代表在多個序列之各對應位置之胺基酸或鹼基的主要選擇。視該系列的多個序列的序列而定，該系列的共有序列與該等序列之各個相差0個、1個、少數個或多個取代。再者，視該系列的多個序列的序列而定，可能為該系列決定超過1個的共有序列。共有序列的產生係經過繁複的數學分析。可以使用各種軟體程式決定共有序列。The term "consensus sequence" as used herein, refers to an amino acid or nucleic acid sequence that is determined by aligning a series of multiple sequences that define an idealized sequence that represents each of a plurality of sequences. The primary choice for the amino acid or base at the corresponding position. Depending on the sequence of the plurality of sequences of the series, the consensus sequence of the series differs from each of the sequences by zero, one, a few or a plurality of substitutions. Furthermore, depending on the sequence of the plurality of sequences in the series, it is possible to determine more than one consensus sequence for the series. The generation of consensus sequences is subject to cumbersome mathematical analysis. A variety of software programs can be used to determine the consensus sequence.

術語「登革熱病毒」或「DEN」係指黃病毒屬及黃病毒科的病毒，其為人類、猴子、黑猩猩及其他動物，尤其是哺乳動物中之登革熱疾病或異常之致病物。術語「登革熱病毒」包括具有四種目前被識別出之血清型：登革熱-1型、登革熱-2型、登革熱-3型及登革熱-4型之任一型登革熱病毒。The term "dengue virus" or "DEN" refers to viruses of the Flavivirus and Flaviviridae family, which are dengue fever diseases or abnormal pathogens in humans, monkeys, chimpanzees and other animals, especially mammals. The term "dengue virus" includes any of the four currently identified serotypes: dengue-1, dengue-2, dengue-3 and dengue-4.

術語「登革熱疾病或異常」用於本文時係指登革熱病毒感染所引起之疾病或異常，其由受感染之蚊子傳染。受感染的蚊子可為埃及斑蚊(Aedes aegypti)(極少為白紋伊蚊(Aedes albopictus))。術語「登革熱疾病或異常」包括登革熱(DF)、出血性登革熱(DHF)、登革熱休克症候群(DSS)以及由登革熱病毒感染所引起之任何其他疾病或異常。The term "dengue disease or abnormality" as used herein refers to a disease or abnormality caused by a dengue virus infection, which is transmitted by an infected mosquito. The infected mosquito can be Aedes aegypti (rarely Aedes albopictus). The term "dengue disease or abnormality" includes dengue fever (DF), hemorrhagic dengue fever (DHF), dengue shock syndrome (DSS), and any other disease or abnormality caused by dengue virus infection.

術語「有效量」用於本文時係指組成物在受治者之組織系統或在研究者、獸醫、醫師或其他臨床人員所研究之受治者中引起生物或醫療反應之量。The term "effective amount" as used herein refers to the amount of a composition that causes a biological or medical response in the subject's tissue system or in a subject studied by a researcher, veterinarian, physician, or other clinical personnel.

在本發明之一具體實施例中，該生物或醫療反應包括由組成物(包含根據本發明之具體實施例之免疫組成物)所誘發的免疫反應。精於本領域人士公知該「免疫有效量」視諸如特定受治者，例如年齡、體重、飲食、健康、先前感染病史 等，以及所用之特定免疫組成物等因素而定。可以進行標準步驟來評估免疫組成物投予至受治者之免疫作用，因此可使精於本技術人士於參照本揭示內容下，決定將被投予至該受治者之根據本發明具體實施例之免疫組成物的免疫有效量。In a particular embodiment of the invention, the biological or medical response comprises an immune response elicited by a composition comprising an immunological composition according to a particular embodiment of the invention. It is well known in the art that the "immunologically effective amount" depends on, for example, a particular subject, such as age, weight, diet, health, history of previous infections. Etc., and the specific immune composition used and other factors. Standard procedures can be performed to assess the immunological effects of administration of the immunological composition to the subject, and thus it will be apparent to those skilled in the art, with reference to the present disclosure, that the invention will be administered to the subject in accordance with the present invention. An immunologically effective amount of an immunological composition.

在本發明之另一具體實施例中，生物或醫療反應包括用根據本發明之具體實施例之疫苗接種所產生之臨床上可觀察到的效益。精於本技術人士公知該「疫苗接種有效量」視諸如特定受治者，例如年齡、體重、飲食、健康、先前感染病史等，以及所用之特定免疫組成物等因素而定。可以進行標準步驟來評估疫苗投予至受治者之免疫作用，因此可使精於本技術人士於參照本揭示內容下，決定將被投予至該受治者之根據本發明具體實施例之疫苗的免疫接種有效量。In another embodiment of the invention, the biological or medical response comprises clinically observable benefits resulting from vaccination according to a particular embodiment of the invention. It is well known to those skilled in the art that the "effective amount of vaccination" depends on factors such as the particular subject, such as age, weight, diet, health, history of previous infections, and the particular immune composition used. Standard procedures can be performed to assess the immunological effects of administration of the vaccine to the subject, and thus it will be apparent to those skilled in the art, with reference to the present disclosure, that a particular embodiment in accordance with the present invention will be administered to the subject. The effective amount of immunization of the vaccine.

在本發明之再一實施例中，生物或醫療反應包括用包括抗體之治療組成物治療登革熱疾病或異常時所產生之臨床上可觀察到之效益，其中該抗體與主要係由序列識別號：5組成之胜肽選擇性結合。根據本發明之一特定實例，該胜肽可為序列識別號：6。在本發明之一具體實施例中，「治療有效量」之根據本發明具體實施例的組成物根除存在的登革熱疾病或異常。在本發明之另一具體實施例中，「治療有效量」之根據本發明具體實施例的組成物於疾病或異常發生之前或之後，將疾病或異常減少至低於當該受治者未接受治療有效量之該組成物時所觀察到者之約10%、20%、30%、40%、50%、60%、70%、80%或90%或更多之程度。可以進行標準步驟來評估將疫苗投予至受治者之療效，此使得精於本技術人士可以參照本揭示內容決定將被投予至該受治者之根據本發明之具體實施例之組成物的治療有效量。In still another embodiment of the invention, the biological or medical response comprises the clinically observable benefits of treating a dengue disease or disorder with a therapeutic composition comprising an antibody, wherein the antibody is identified by a sequence identifier: 5 consists of a peptide that selectively binds. According to a particular embodiment of the invention, the peptide can be a sequence identifier: 6. In a particular embodiment of the invention, a "therapeutically effective amount" of a composition according to a particular embodiment of the invention eradicates the presence of dengue disease or abnormality. In another embodiment of the invention, a "therapeutically effective amount" of a composition according to a particular embodiment of the invention reduces the disease or abnormality below or after the disease or abnormality occurs when the subject does not receive The therapeutically effective amount of the composition is about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% or more as observed. Standard procedures can be performed to assess the efficacy of administering a vaccine to a subject, which enables a person skilled in the art to determine, in light of the present disclosure, a composition according to a particular embodiment of the present invention to be administered to the subject. The therapeutically effective amount.

術語「表位(epitope)」用於本文時係指抗原與單一性抗體分子或T-細胞受體結合之位點。「表位」用於本文時，可 與「抗原決定子(antigenic determinant)」或「抗原決定位點(antigenic determinant site)」交互使用。The term "epitope" as used herein refers to a site at which an antigen binds to a single antibody molecule or a T-cell receptor. When "epitope" is used in this article, Interact with "antigenic determinant" or "antigenic determinant site".

術語「免疫反應(immune response)」或「產生免疫力反應(immuogenic response)」用於本文時係指在受治者中免疫系統回應抗原之任何反應。在脊椎動物中免疫反應之例子包括，但不限於，抗體製造、細胞媒介之免疫力之誘生、補體活化及免疫耐受性之生成。對於相同抗原所引起之接續刺激之免疫反應，亦稱為繼發性免疫反應，其產生較原發性免疫反應之情況快速。The term "immune response" or "immuogenic response" as used herein refers to any response of the immune system to an antigen in a subject. Examples of immune responses in vertebrates include, but are not limited to, antibody production, induction of immunity by cellular media, complement activation, and production of immune tolerance. The immune response to subsequent stimuli caused by the same antigen, also known as the secondary immune response, produces a faster response than the primary immune response.

本發明實質上經純化之單離、重組或合成胜肽，即為實質上不含來自衍生該胜肽之細胞或組織來源之細胞物質或其他污染性胜肽或蛋白質之胜肽，或者當化學合成時實質上不含化學先質或其他化學品之胜肽。實質上不含細胞物質之胜肽之製劑包括：所含之胜肽與該經單離胜肽所來自之細胞或重組製造該胜肽之細胞的細胞成分分離者。因此，實質上不含細胞物質之胜肽包括：具有少於約30%、20%、10%或5%(以重量計)之異源蛋白質或胜肽(在本文中亦稱為「污染性蛋白質或胜肽」)之胜肽的製劑。當該胜肽被重組製造時，亦以實質上不含培養基為較佳，即培養基)於該胜肽製劑之體積之約20%、10%或5%。當該胜肽藉由化學合成法製造時，較佳實質上不含化學先質或其他化學品，亦即其與涉及該胜肽合成之化學先質或其他化學品分離。所以，該胜肽之製劑具有少於約30%、20%、10%、5%(以重量計)之該感興趣胜肽以外之化學先質或化合物。尤其是，但非絕對地，當將單離、重組或合成胜肽之製劑投予至受治者以供疫苗接種或其他醫藥目的使用時，該製劑只可含有對於該受治者不具生物不良作用之量之污染胜肽或蛋白質、其他細胞物質或其他化學品。The substantially purified, recombinant or synthetic peptide of the present invention is substantially free of cellular material or other contaminating peptide or protein derived from the cell or tissue from which the peptide is derived, or as a chemical A peptide that is substantially free of chemical precursors or other chemicals when synthesized. The preparation of the peptide which is substantially free of cellular material includes a peptide which is separated from the cell from which the isolated peptide is derived or which is recombinantly produced. Thus, a peptide that is substantially free of cellular material includes: less than about 30%, 20%, 10%, or 5% by weight of a heterologous protein or peptide (also referred to herein as "contaminating" A preparation of a peptide of a protein or peptide "). When the peptide is produced recombinantly, it is preferably substantially free of the medium, i.e., the medium, about 20%, 10% or 5% of the volume of the peptide preparation. When the peptide is produced by chemical synthesis, it is preferably substantially free of chemical precursors or other chemicals, i.e., it is separated from chemical precursors or other chemicals involved in the synthesis of the peptide. Thus, the peptide preparation has less than about 30%, 20%, 10%, 5% by weight of a chemical precursor or compound other than the peptide of interest. In particular, but not exclusively, when a formulation of an isolated, recombinant or synthetic peptide is administered to a subject for vaccination or other medical purposes, the formulation may only contain no biological defects for the subject The amount of action is a peptide or protein, other cellular material, or other chemical.

該單離、重組或合成胜肽可具有數種不同的物理形式。該胜肽可以全長、原生(nascent)或未經加工的胜肽，或以經部分加工的胜肽或以經加工胜肽之組合存在。全長原生胜肽可藉由能造成全長原生胜肽之片段生成之單一性蛋白酶裂解事件而加以轉譯後修飾。該胜肽可藉由單離核酸序列來編碼或者藉由，例如，化學合成方法而合成。可將該胜肽與生物物質、化學先質或其他化學品分離，然後使用習知蛋白質分析或製備步驟將其單離至得以根據本文所述之方法使用之程度。The isolated, recombinant or synthetic peptide can have several different physical forms. The peptide can be present in full length, nascent or unprocessed peptides, or as a partially processed peptide or as a combination of processed peptides. The full length native peptide can be post-translationally modified by a single protease cleavage event that results in a fragment of the full length native peptide. The peptide can be encoded by a single nucleic acid sequence or by, for example, a chemical synthesis method. The peptide can be separated from the biological material, chemical precursor or other chemical and then isolated using a conventional protein analysis or preparation step to the extent that it can be used according to the methods described herein.

在本文中，術語「中和」當用於敘述抗體反應時，係指抗體與登革熱病毒之一位點結合以阻斷登革熱病毒停泊或與宿主細胞結合，因此可防止該宿主細胞被登革熱病毒感染之能力。登革熱病毒利用該位點阻斷其本身與宿主細胞上之受體結合，以致不會感染該宿主細胞。本文中所用之術語「交叉中和」係指抗體與登革熱病毒之多種血清型結合，以阻斷該等病毒停泊或與宿主細胞結合，因而防止該宿主細胞被多種血清型登革熱病毒感染。As used herein, the term "neutralization" when used to describe an antibody response means that the antibody binds to one of the sites of the dengue virus to block dengue virus berthing or binding to the host cell, thereby preventing the host cell from being infected by the dengue virus. Ability. The dengue virus uses this site to block its own binding to receptors on the host cell so that it does not infect the host cell. As used herein, the term "cross-neutralization" refers to the binding of an antibody to various serotypes of dengue virus to block the docking or binding of the virus to the host cell, thereby preventing the host cell from being infected with a plurality of serotype dengue viruses.

術語「胜肽抗原」用於本文時係指由胜肽所組成之抗原。「胜肽抗原」含有1個或多個抗原決定位點，各由單一性的胺基酸序列形成。該胜肽抗原可包括保留該抗原之免疫單一性之胺基酸取代。The term "peptide antigen" as used herein refers to an antigen consisting of a peptide. A "peptide antigen" contains one or more epitopes, each formed by a single amino acid sequence. The peptide antigen can include an amino acid substitution that retains the immunomonotropy of the antigen.

「胜肽」係指排列成聚合物之胺基酸殘基。胜肽可由標準的20個天然胺基酸組成，此外可含有罕見胺基酸及合成胺基酸類似物。「重組胜肽」係指由重組DNA技術產生之胜肽，即從藉由編碼該期望胜肽之外來DNA構築體所轉形之細胞產生之胜肽。「合成胜肽」係指藉由化學合成製備者。"Peptide" means an amino acid residue arranged in a polymer. The peptide can be composed of standard 20 natural amino acids, and can also contain rare amino acids and synthetic amino acid analogs. "Recombinant peptide" refers to a peptide produced by recombinant DNA technology, that is, a peptide produced by a cell transformed by a DNA construct other than the desired peptide. "Synthetic peptide" means a preparation prepared by chemical synthesis.

術語「受治者或受檢者(subject)」用於本文時，係指為了診斷、治療、觀察或實驗之目標之動物，較佳為哺乳動物。 該受治者或受檢者之例子為人類、飼養動物(肉牛及乳牛、綿羊、家禽及豬等)，或陪伴動物(狗、貓、馬等)。The term "subject" or "subject" as used herein refers to an animal, preferably a mammal, for the purpose of diagnosis, treatment, observation or experimentation. Examples of the subject or subject are humans, animals (beef cattle and cows, sheep, poultry, pigs, etc.), or companion animals (dogs, cats, horses, etc.).

術語「治療登革熱疾病或異常」，或「登革熱疾病或異常之治療」意指制止、緩和、減輕、減少疾病或預防登革熱疾病或異常。「治療登革熱疾病或異常」包括存在之登革熱疾病或異常之治療性處理以及在登革熱疾病或異常之症狀浮現或可觀察到之前之預防性處理二者。「治療登革熱疾病或異常」產生臨床上可觀察到之效益。臨床上可觀察到之效益包括一種治療性處理狀態，其中於存在之登革熱疾病或異常之症狀可觀察到後將本發明之組成物投予至受治者，以致該存在之登革熱疾病或異常之可觀察到症狀不是根除就是減輕至低於未治療者之程度。臨床上可觀察到之效益亦包括一種預防性處理狀態，其中於登革熱疾病或異常之症狀可觀察到之前，防止登革熱疾病或異常之症狀發生，或者雖然登革熱疾病或異常之症狀隨後發生，但其程度比未投予本發明之組成物者輕。The term "treating dengue disease or abnormality" or "treatment of dengue fever or abnormality" means stopping, mitigating, reducing, reducing or preventing dengue fever or abnormalities. "Treatment of dengue disease or abnormalities" includes both the presence of dengue disease or aberrant therapeutic treatment and the presence of dengue fever or abnormal symptoms or pre-existing prophylactic treatment. "Treatment of dengue fever or abnormalities" produces clinically observable benefits. A clinically observable benefit includes a therapeutic treatment state in which a composition of the present invention is administered to a subject after the presence of a dengue disease or abnormal condition, such that the dengue disease or abnormality is present It can be observed that the symptoms are not eradicated or reduced to a lesser extent than untreated. Clinically observable benefits also include a prophylactic treatment state in which dengue fever disease or abnormal symptoms are observed before prevention of dengue fever or abnormal symptoms, or although dengue fever or abnormal symptoms subsequently occur, but To a lesser extent than those not administered to the compositions of the present invention.

術語「疫苗」用於本文時，係指意欲投予至受治者以在受治者體內產生或人為增加對特定疾病之免疫反應的任何製劑。疫苗之例子包括殺死之感染物(常為毒性株)之製劑，該等感染物包括，但不限於，微生物(細菌、病毒等)、真菌、原生動物、後生動物(metazoan)或植物。疫苗之例子亦包括活感染物之製劑，常為減毒(變異或突變)株之製劑。疫苗之例子進一步包括感染物之次組成分(sub-parts)、部分(portions)或產物，諸如得自感染物之胜肽抗原。The term "vaccine" as used herein, refers to any preparation intended to be administered to a subject to produce or artificially increase the immune response to a particular disease in the subject. Examples of vaccines include preparations of killed infectious agents (often virulence strains) including, but not limited to, microorganisms (bacteria, viruses, etc.), fungi, protozoa, metazoans or plants. Examples of vaccines also include preparations of live infectious agents, often in the form of attenuated (variant or mutant) strains. Examples of vaccines further include sub-parts, parts or products of the infectious agent, such as peptides derived from the infectious agent.

在成熟登革熱病毒粒子中存在有三種結構蛋白質：核心核衣殼蛋白質(core nucleocapsid protein)(C)、跨膜蛋白質(M)及主要包膜糖蛋白(E)。來自登革熱病毒及其他黃病毒之E蛋白質之結晶研究顯示其含有三種結構域(I-III)(Modis et al., 2003,PNAS 100: 6986-6991; Modis et al., 20050J. Virol. 79:1223-1231; Rey et al.,Nature 375: 291-298)。E蛋白質之第三區塊具有IgG-樣摺疊部及參與大多數相關生物事件(包括毒性及/或神經毒性)之決定位點(Cecilia et al., 1991, Virology 181:70-77; Leitmeyer et al., 1999,J. Virol. 73:4738-4747; Lobigs et al., 1990, Virology 176:587-595; Pletnev et al., 1993, J.Virol. 67:4956-4963; Sanchez et al., 1996,J Gen Virol 77:2541-2545; Sumiyoshi et al., 1995,J. Infect Dis 171:1144-1151)。第三區塊亦涉及與宿主受體之結合(Chen et al., 1997,Nat Med 3:866-871; and Hung et al., 2004,J. Virol. 78:378-3883)且其含有只會誘生出中和抗體之類型及亞型-單一性抗原決定位點(Crill et al., 2001,J. Virol. 75:7769-7773; Hiramatsu et al., 1996,Virology 224: 437-445; Roehrig, et al.,1998,Virology 246:317-328; Roehrig et al., 1994,Virology 198:31-38; Thullier et al., 2001,J. Gen Virol 82:1885-1892; Trirawatanapong et al., 1992.Gene 116:139-150)。縱使包膜蛋白之第三區塊(E3)僅代表包膜蛋白之一部分，但誘生出非中和、交叉反應性抗體之其他表位(epitope)之不存在將具有減少惡化成DHF或DSS之風險之優點。已證明在小鼠模型中用編碼E3之DNA進行免疫會誘生出對抗登革熱病毒之保護性抗體(Mota et al., 2005, Vaccine 23:3469-3476)。此等結果暗示E3為有潛力的亞單位疫苗候選者。There are three structural proteins in the mature dengue virus particles: core nucleocapsid protein (C), transmembrane protein (M) and major envelope glycoprotein (E). Crystallization of E proteins from dengue virus and other flaviviruses revealed three domains (I-III) (Modis et al., 2003, PNAS 100: 6986-6991; Modis et al., 20050 J. Virol. 79) :1223-1231; Rey et al., Nature 375: 291-298). The third block of E protein has an IgG-like fold and a determinant site involved in most related biological events, including toxicity and/or neurotoxicity (Cecilia et al., 1991, Virology 181:70-77; Leitmeyer et Al., 1999, J. Virol. 73:4738-4747; Lobigs et al., 1990, V irology 176:587-595; Pletnev et al., 1993, J. Virol. 67:4956-4963; Sanchez et al 1996, J Gen Virol 77: 2541-2545; Sumiyoshi et al., 1995, J. Infect Dis 171:1144-1151). The third block also involves binding to host receptors (Chen et al., 1997, Nat Med 3: 866-871; and Hung et al., 2004, J. Virol. 78:378-3883) and it contains only Types of neutralizing antibodies and subtype-single epitopes are induced (Crill et al., 2001, J. Virol. 75:7769-7773; Hiramatsu et al., 1996, Virology 224: 437-445; Roehrig, et al., 1998, Virology 246:317-328; Roehrig et al., 1994, Virology 198:31-38; Thullier et al., 2001, J. Gen Virol 82:1885-1892; Trirawatanapong et al. , 1992. Gene 116: 139-150). Even if the third block of envelope protein (E3) represents only one part of the envelope protein, the absence of other epitopes that induce non-neutralizing, cross-reactive antibodies will have a reduction in degradation to DHF or DSS. The advantages of risk. Immunization with DNA encoding E3 in a mouse model has been shown to induce protective antibodies against dengue virus (Mota et al., 2005, Vaccine 23: 3469-3476). These results suggest that E3 is a potential subunit vaccine candidate.

根據本發明從四種血清型登革熱病毒之E3設計共有胜肽。該共有胜肽能誘生中和抗體反應，對抗多種血清型登革熱病毒。The consensus peptide was designed from E3 of four serotypes of dengue virus according to the present invention. The consensus peptide is capable of inducing a neutralizing antibody response against a variety of serotype dengue viruses.

在一具體實施例中，本發明係關於主要係由序列識別號：5組成之單離、重組或合成胜肽。序列識別號：5係藉由來自登革熱病毒之四種血清型之E3的序列分析而設計成的共有胜肽序列。根據一特定實施例，合成胜肽主要係由序列識別號：6組成。序列識別號：6為從序列識別號：5之位置7 至40衍生的合成胜肽序列。可使用由序列識別號：5或序列識別號：6組成之胜肽作為胜肽抗原，誘生出對抗登革熱病毒之一種或多種或較佳所有四種血清型的免疫反應。In a specific embodiment, the invention relates to isolated, recombinant or synthetic peptides consisting essentially of sequence identifier:5. SEQ ID NO: 5 is a consensus peptide sequence designed by sequence analysis of E3 from four serotypes of dengue virus. According to a particular embodiment, the synthetic peptide consists essentially of the sequence identifier: 6. Sequence identification number: 6 is from the sequence identification number: 5 position 7 Synthetic peptide sequence derived to 40. A peptide consisting of SEQ ID NO: 5 or SEQ ID NO: 6 can be used as a peptide antigen to induce an immune response against one or more or preferably all four serotypes of the dengue virus.

主要係由序列識別號：5組成之胜肽可藉由各種方式製造。例如，胜肽可藉由重組DNA技術或從試管內轉譯系統製造，或者使用標準的胜肽合成技術而化學合成。在較佳具體實施例中，該胜肽藉由化學合成法諸如在自動胜肽合成器上進行固相胜肽合成而製造。該方法為精於本技術者所已知且易根據本揭示內容而調整成適用於本發明。The peptide consisting mainly of the sequence identification number: 5 can be produced by various means. For example, peptides can be made by recombinant DNA techniques or from in-tube translation systems, or chemically synthesized using standard peptide synthesis techniques. In a preferred embodiment, the peptide is produced by chemical synthesis, such as solid phase peptide synthesis on an automated peptide synthesizer. This method is known to those skilled in the art and is readily adapted to the present invention in light of the present disclosure.

主要係由序列識別號：5組成之胜肽可參照本揭示內容藉由精於本技術者已知之方法實質純化。例如，胜肽可藉由鹽分分離(salt fractionation)、離子交換層析、分子大小排除層析、羥磷灰石吸附層析及疏水***互作用層析、凝集素層析、HPLC及FPLC、以及抗體/配體親核性層析之各種組合或個別應用而從細胞溶裂物及萃取物或從化學合成之反應混合物實質純化。The peptide consisting essentially of the sequence identifier: 5 can be substantially purified by reference to the disclosure by methods well known to those skilled in the art. For example, the peptide can be separated by salt fractionation, ion exchange chromatography, molecular size exclusion chromatography, hydroxyapatite adsorption chromatography and hydrophobic interaction chromatography, lectin chromatography, HPLC and FPLC, and Substantially purified from cell lysates and extracts or from chemically synthesized reaction mixtures in various combinations or individual applications of antibody/ligand nucleophilic chromatography.

在另一具體實施例中，本發明係關於包括主要係由序列識別號：5組成之胜肽之免疫組成物。該免疫組成物包括主要係由序列識別號：5組成之胜肽，其量為當將該免疫組成物投予至受治者時能有效誘發免疫反應之量。在一些具體實施例中，該免疫組成物每投予劑量包括約0.5微克(μg)至約1毫克(mg)主要係由序列識別號：5組成之胜肽。例如，根據本發明具體實施例之免疫組成物每投予劑量包括約0.5微克至10微克，約10微克至100微克，約100微克至500微克或約500微克至1000微克之主要係由序列識別號：5組成之胜肽。In another specific embodiment, the invention relates to an immunological composition comprising a peptide consisting essentially of SEQ ID NO: 5. The immunological composition includes a peptide consisting essentially of SEQ ID NO: 5 in an amount effective to elicit an immune response when the immunological composition is administered to a subject. In some embodiments, the immunological composition comprises from about 0.5 micrograms ([mu]g) to about 1 milligram (mg) of a peptide consisting essentially of SEQ ID NO: 5 per dose administered. For example, an immunological composition according to a specific embodiment of the present invention comprises from about 0.5 microgram to 10 micrograms per dose, from about 10 micrograms to 100 micrograms, and from about 100 micrograms to 500 micrograms or from about 500 micrograms to about 1000 micrograms. No.: 5 consists of a peptide.

除了主要係由序列識別號：5組成之胜肽之外，該免疫組成物亦包括其他成分，諸如生理可接受之載劑及/或佐劑。 該載劑及/或佐劑為該領域之已知技術。該載劑包括，但不限於，水、緩衝水、食鹽溶液、甘胺酸溶液、透明質酸及類似物、pH調整及緩衝劑、張力調整劑及潤濕劑。可用於本發明之佐劑之非限定例子述於下文。In addition to the peptide consisting essentially of SEQ ID NO: 5, the immunological composition also includes other components such as physiologically acceptable carriers and/or adjuvants. Such carriers and/or adjuvants are well known in the art. Such carriers include, but are not limited to, water, buffered water, saline solutions, glycine acid solutions, hyaluronic acid and the like, pH adjusting and buffering agents, tonicity adjusting agents, and wetting agents. Non-limiting examples of adjuvants that can be used in the present invention are described below.

在另一具體實施例中，本發明係關於一種套組，其包括：根據本發明之具體實施例之免疫組成物以及說明在受治者中使用該免疫組成物誘發免疫反應之說明書。例如，該套組可為含有該組成物之容器，諸如瓶子、廣口瓶或盒子，連同附於容器上或含於容器內(諸如固定或印刷於容器之表面或標籤上)之說明書，。In another embodiment, the invention is directed to a kit comprising: an immunological composition according to a particular embodiment of the invention and instructions for inducing an immune response using the immunological composition in a subject. For example, the kit can be a container containing the composition, such as a bottle, jar or box, along with instructions attached to or contained within the container, such as on a surface or label of the container.

本發明之另一具體實施例亦包括在受治者中誘發免疫反應之方法。該方法包括將免疫有效量之根據本發明之任一具體實施例之免疫組成物投予至受治者。該免疫組成物可藉由各種方式，諸如攝入、鼻噴霧、皮下注射(SC)、腹膜腔內注射(IP)、靜脈輸注、靜脈內注射、肌肉內注射、透皮貼片或其組合而投予至受治者。免疫反應包括對抗登革熱病毒之一種或多種血清型之中和抗體反應。免疫反應較佳包括對抗所有四種血清型登革熱病毒之中和抗體反應。Another embodiment of the invention also includes a method of inducing an immune response in a subject. The method comprises administering an immunologically effective amount of an immunological composition according to any of the embodiments of the present invention to a subject. The immunological composition can be administered by various means such as ingestion, nasal spray, subcutaneous injection (SC), intraperitoneal injection (IP), intravenous infusion, intravenous injection, intramuscular injection, transdermal patch or a combination thereof. Invested in the subject. The immune response involves neutralizing antibody responses against one or more serotypes of dengue virus. Preferably, the immune response comprises neutralizing antibody responses against all four serotypes of dengue virus.

在本發明之特定具體實施例中，該免疫組成物為用於對抗選自血清型1至4登革熱病毒所組成之群中之一種或多種登革熱病毒的疫苗。該疫苗在受治者中可用於預防登革熱疾病或異常。在一具體實施例中，該疫苗用於預防嚴重或危及生命的登革熱疾病諸如DHF或DSS。將疫苗接種有效量之根據本發明之具體實施例之疫苗以單次投予或多次投予之方式投予至受治者。可能需要多次投予疫苗以引起足夠程度之免疫力。誘發免疫程度之監測可藉由測量來自受治者之生物檢體之中和抗體量。需要時可調整疫苗接種劑量及次數以維持所需的保護程度。In a particular embodiment of the invention, the immunological composition is a vaccine for combating one or more dengue viruses selected from the group consisting of serotypes 1 to 4 dengue viruses. The vaccine can be used to prevent dengue disease or abnormalities in the subject. In a specific embodiment, the vaccine is used to prevent severe or life-threatening dengue diseases such as DHF or DSS. The vaccination effective amount of the vaccine according to the specific embodiment of the present invention is administered to the subject in a single administration or multiple administration. It may be necessary to administer the vaccine multiple times to induce a sufficient level of immunity. Monitoring of the degree of induced immunity can be achieved by measuring the amount of antibody in the biosample from the subject. The vaccination dose and frequency can be adjusted as needed to maintain the desired level of protection.

在本發明之某些具體實施例中，可單獨投予本發明具體實施例之疫苗，或與可誘發保護反應對抗一或多種其他感染物或藉由其他作用機制對抗之一或多種血清型登革熱病毒的一或多種其他疫苗一起投予。一起投予涵蓋在投予本發明具體實施例之疫苗之同時、之前或之後所投予的其他疫苗。在本領域中具有普通技術之人士於參照本揭示內容下，可輕易地決定投予特定疫苗之適當時機、順序及劑量。In certain embodiments of the invention, the vaccine of a particular embodiment of the invention may be administered alone, or may be induced to counteract one or more other infectious agents or by other mechanisms of action against one or more serotypes of dengue One or more other vaccines of the virus are administered together. The other vaccines administered at the same time, before or after administration of the vaccine of the specific embodiment of the present invention are administered together. Those of ordinary skill in the art, with reference to the present disclosure, can readily determine the appropriate timing, sequence, and dosage for administering a particular vaccine.

本發明之另一廣義方面係關於選擇性與主要係由序列識別號：5組成之胜肽結合之經實質純化的抗體。根據本發明之具體實施例之抗體單一性與登革熱病毒之一種或多種血清型的包膜蛋白結合。該抗體係單一性與該包膜蛋白之第三區塊，更特定而言，為第三區塊中與序列識別號：5實質相似的序列結合。Another broad aspect of the invention pertains to substantially purified antibodies that bind selectively to a peptide consisting essentially of the sequence ID:5. The antibody singularity according to a particular embodiment of the invention binds to an envelope protein of one or more serotypes of dengue virus. The anti-system singularity is combined with a third block of the envelope protein, more specifically, a sequence substantially similar to SEQ ID NO: 5 in the third block.

根據本發明之具體實施例之抗體可藉由各種方法製造。在一具體實施例中，多株抗體可藉由使用本發明之免疫原(連帶或未連帶免疫佐劑)使適當的受治動物諸如小鼠、老鼠、天竺鼠、兔子、山羊、馬及類似動物(以兔子為較佳)免疫而產生。在第一次進行免疫之前收集免疫前的血清。各動物接受約0.001mg與約1000mg之間的免疫原，該免疫原與或未與可接受的免疫佐劑締合。初始免疫可用主要係由序列識別號：5組成之胜肽進行，該胜肽較佳在弗氏完全佐劑中且經皮下(SC)或腹膜腔內(IP)或二者在多個位點投予。每隔一定時間，較佳每星期，將各動物放血以測定抗體滴度。於初始免疫之後，此等動物可接受或不接受追加注射。此等接受追加注射之動物通常係經由相同途徑接受等量之在弗氏不完全佐劑中之抗原。每隔約3星期給予追加注射直至得到最大滴度。於各次追加免疫後約7天或於單次注射後約每星期，將動物放血，收集血清及將等分試樣貯存於約-20℃。Antibodies according to particular embodiments of the invention can be made by a variety of methods. In a specific embodiment, a plurality of antibodies can be administered to an appropriately treated animal such as a mouse, a mouse, a guinea pig, a rabbit, a goat, a horse, and the like by using the immunogen of the present invention (with or without an immunoadjuvant). Produced by immunization (preferred by rabbits). Pre-immune serum was collected prior to the first immunization. Each animal receives between about 0.001 mg and about 1000 mg of immunogen associated with or not associated with an acceptable immunological adjuvant. The initial immunization can be performed mainly by a peptide consisting of SEQ ID NO: 5, which is preferably in Freund's complete adjuvant and subcutaneously (SC) or intraperitoneal (IP) or both at multiple sites. Cast. Each animal is bled at regular intervals, preferably weekly, to determine antibody titers. These animals may or may not receive additional injections after initial immunization. Such animals receiving additional injections typically receive an equal amount of antigen in Freund's incomplete adjuvant via the same route. Additional injections were given every 3 weeks until the maximum titer was obtained. The animals were bled about 7 days after each booster immunization or about once a week after a single injection, serum was collected and aliquots were stored at about -20 °C.

在另一具體實施例中，單株抗體可藉由使用本發明之免疫原將養殖的小鼠(較佳Balb/c)免疫而製備。如上文所討論，該小鼠藉由使用約0.001mg至約1.0mg，較佳地約0.1mg之免疫原經由IP或SC途徑加以免疫，其中該免疫原係在納入等量可接受佐劑之約0.1ml緩衝液或食鹽水中。佐劑較佳為弗氏佐劑，其中弗氏完全佐劑用於初始免疫，弗氏不完全佐劑則於隨後使用。小鼠於第0日接受初始免疫，隨後休息約2至約30星期。給予已免疫小鼠1次或多次追加免疫，該追加免疫係使用在緩衝溶液(諸如磷酸鹽緩衝食鹽水)中之約0.001mg至約1.0mg之主要係由序列識別號：5組成之胜肽經由靜脈內注射(IV)途徑而達成。In another embodiment, a monoclonal antibody can be prepared by immunizing a cultured mouse (preferably Balb/c) using the immunogen of the invention. As discussed above, the mouse is immunized via an IP or SC route by using from about 0.001 mg to about 1.0 mg, preferably about 0.1 mg of the immunogen, wherein the immunogen is in an equivalent amount of an acceptable adjuvant. About 0.1 ml of buffer or saline. The adjuvant is preferably Freund's adjuvant, wherein Freund's complete adjuvant is used for initial immunization and Freund's incomplete adjuvant is used subsequently. Mice received initial immunization on day 0 followed by a rest of about 2 to about 30 weeks. The immunized mouse is given one or more additional immunizations, and the booster is used in a buffer solution (such as phosphate buffered saline) from about 0.001 mg to about 1.0 mg, which is mainly composed of sequence identification number: 5 Peptides are achieved via the intravenous (IV) route.

來自抗體陽性小鼠之淋巴球(以脾臟淋巴球為較佳)，藉由以本領域已知之標準步驟從經免疫之小鼠移出脾臟而得到。融合瘤細胞係藉由將脾臟淋巴球與適當的融合配偶(以骨髓瘤細胞為較佳)於允許安定融合瘤形成之條件下加以混合而產生。融合配偶可包括，但不限於，小鼠骨髓瘤P3/NS1/Ag4-1、MPC-11、S-194及Sp2/0，其中通常以Sp2/0為較佳。將產生抗體之細胞與骨髓瘤細胞在分子量約1000聚乙二醇中，以約30%至約50%之濃度進行融合。經融合而得的融合瘤細胞於參照本揭示內容下，藉由本領域已知之步驟在補充有次黃嘌呤、胸腺嘧啶及胺基嘌呤之經杜百可修飾之伊格培養基(DMEM)中培養而加以選擇。於約第14、18及21天從生長陽性孔收集上清液，以及使用主要係由序列識別號：5組成之胜肽作為抗原藉由免疫分析法諸如固相免疫放射分析(SPIRA)而篩選抗體的產生。該培養液亦在Ouchterlony沉澱分析中被測試，以測定mAb之同型(isotype)。Lymphocytes from antibody-positive mice (preferably spleen lymphocytes) are obtained by removing the spleen from the immunized mice by standard procedures known in the art. The fusion tumor cell line is produced by mixing the spleen lymphocytes with a suitable fusion partner (preferably myeloma cells) under conditions that permit stable fusion formation. Fusion partners may include, but are not limited to, mouse myeloma P3/NS1/Ag4-1, MPC-11, S-194, and Sp2/0, with Sp2/0 being preferred. The antibody-producing cells are fused with myeloma cells in a molecular weight of about 1000 polyethylene glycol at a concentration of from about 30% to about 50%. The fused fusion tumor cells are cultured in a Dopco's modified Ig medium (DMEM) supplemented with hypoxanthine, thymine, and amine guanidine by reference to the present disclosure by procedures known in the art. Choose it. The supernatant was collected from growth-positive wells on days 14, 18 and 21, and the peptides consisting mainly of SEQ ID NO: 5 were used as antigens by immunoassay such as solid phase immunoradiometric assay (SPIRA). Production of antibodies. This culture was also tested in the Ouchterlony precipitation assay to determine the isotype of the mAb.

來自抗體陽性孔之融合瘤細胞係藉由已知技術諸如MacPherson之軟瓊脂技術(Soft Agar Techniques, in Tissue Culture Methods and Applicationns, Kruse and Paterson, Eds., Academic Press, 1973)或藉由有限稀釋之技術而進行選殖。單株抗體係藉由於促發(priming)後至少約4日，以每隻小鼠約0.5ml(含約1x106 至約6x106 個融合瘤細胞)之量注射至以降植烷(pristine)促發之Balb/c小鼠而在活體內產生。於細胞轉移後約8至12日收集腹水以及藉由本領域已知之技術純化單株抗體。單株抗體亦可於參照本揭示內容下，藉由在本領域熟知之組織培養基中培養融合瘤而在試管內產生。可使用本領域熟知之中空纖維培養技術、氣升式反應器、輥瓶或旋轉燒瓶培養技術進行高密度試管內細胞培養，以產生大量的mAbs。該等mAbs係於參照本揭示內容下，藉由在本領域已知之技術純化。Fusion cell lines derived from antibody positive wells are known by techniques such as Soft Agar Techniques, in Tissue Culture Methods and Applicationns, Kruse and Paterson, Eds., Academic Press, 1973, or by limiting dilution. Cultivation is carried out by technology. Monoclonal antibody is an amount by precipitating after (priming) of at least about 4 days to about 0.5ml per mouse (containing from about 1x10 6 to about 6x10 6 integrates both tumor cells) injected into the phytane to reduce (Pristine) pro It is produced in vivo by Balb/c mice. Ascites was collected approximately 8 to 12 days after cell transfer and the monoclonal antibodies were purified by techniques known in the art. Monoclonal antibodies can also be produced in vitro by culturing a fusion tumor in a tissue culture medium well known in the art, with reference to the present disclosure. High density in vitro cell culture can be performed using hollow fiber culture techniques well known in the art, airlift reactors, roller bottles or spinner flask culture techniques to produce large amounts of mAbs. Such mAbs are purified by techniques known in the art with reference to the present disclosure.

含有根據本發明之具體實施例之抗體之製劑之滴度可藉由各種血清學或免疫學分析而測定。此等分析包括，但不限於，沉澱、被動凝集、酶-鍵結免疫吸附抗體(ELISA)技術及放射免疫分析(RIA)技術。The titer of a formulation containing an antibody according to a particular embodiment of the invention can be determined by various serological or immunological assays. Such assays include, but are not limited to, precipitation, passive agglutination, enzyme-binding immunosorbent antibody (ELISA) techniques, and radioimmunoassay (RIA) techniques.

參照本發明揭示內容，本發明具體實施例之抗體可從哺乳動物(例如從血液)或培養細胞單離以及進一步藉由熟知的技術純化。例如，可使用蛋白質A層析得到IgG部分。或者，對本發明之胜肽具單一性之抗體可藉由例如親和性層析而選擇(例如部分純化)或純化。例如，主要係由序列識別號：5組成之經單離或經實質純化之胜肽如本文所述製造，且被共價地或非共價地偶合至固相支持物，諸如，例如親和層析管柱。然後在含有對抗大量不同表位之抗體的檢體，可使用該管柱對本發明具體實施例之胜肽具單一性的抗體進行親和性純化，藉此產生經實質純化之抗體組成物，即實質不含污染抗體者。With reference to the present disclosure, antibodies of particular embodiments of the invention can be isolated from mammalian (e.g., from blood) or cultured cells and further purified by well known techniques. For example, protein A can be used to obtain an IgG fraction. Alternatively, an antibody that is singular to the peptide of the present invention can be selected (e.g., partially purified) or purified by, for example, affinity chromatography. For example, an isolated or substantially purified peptide consisting essentially of SEQ ID NO: 5 is made as described herein and is covalently or non-covalently coupled to a solid support, such as, for example, an affinity layer. Pipe column. Then, in a sample containing an antibody against a large number of different epitopes, the column can be used to affinity-purify the antibody having a single peptide of the specific embodiment of the present invention, thereby producing a substantially purified antibody composition, that is, the substance. Contains no contaminating antibodies.

此外，可用標準重組DNA技術製造之重組抗體，諸如嵌合及人型化單株抗體(包括人類及非人類部分二者)，在本發明之範圍內。嵌合抗體為分子中不同部分衍生自不同動物 物種者，諸如具有衍生自鼠mAb之可變區及人類免疫球蛋白恆定區者。人型化抗體為來自非人類物種之抗體分子，其具有1個或多個來自非人類物種之互補決定區(CDRs)及來自人類免疫球蛋白分子之框架區。此等嵌合及人型化單株抗體可藉由本領域已知之重組DNA技術製造。Furthermore, recombinant antibodies, such as chimeric and humanized monoclonal antibodies (both human and non-human), which can be made using standard recombinant DNA techniques, are within the scope of the invention. Chimeric antibodies are derived from different animals in different parts of the molecule Species, such as those having a variable region derived from a murine mAb and a human immunoglobulin constant region. A humanized antibody is an antibody molecule from a non-human species having one or more complementarity determining regions (CDRs) from a non-human species and a framework region from a human immunoglobulin molecule. Such chimeric and humanized monoclonal antibodies can be made by recombinant DNA techniques known in the art.

完全的人類抗體對於人類病患之治療性處理而言特別受到期待。此等抗體，例如，可藉由使用無法表現內源免疫球蛋白重鏈及輕鏈基因，但可表現人類重鏈及輕鏈基因的基因轉移小鼠製造。將該基因轉移小鼠以所選的抗原，例如本發明胜肽之全部或一部分，以正常方式予以免疫。對抗該抗原之單株抗體可藉由使用習知的融合瘤技術而得到。停泊在基因轉移小鼠中之人類免疫球蛋白轉移基因於B細胞分化期間重排，接下來進行類別轉換及體細胞突變。因此，使用該技術可以產生治療上有用的IgG、IgA及IgE抗體。Complete human antibodies are particularly expected for the therapeutic treatment of human patients. Such antibodies can be produced, for example, by using a gene transfer mouse that does not express endogenous immunoglobulin heavy and light chain genes but can express human heavy and light chain genes. The gene is transferred to a mouse and immunized in a normal manner with a selected antigen, such as all or a portion of the peptide of the present invention. A monoclonal antibody against the antigen can be obtained by using a conventional fusion tumor technique. The human immunoglobulin transfer gene, which is parked in gene transfer mice, rearranges during B cell differentiation, followed by class switching and somatic mutation. Thus, therapeutically useful IgG, IgA, and IgE antibodies can be produced using this technique.

能識別所選表位之完全人類抗體可用稱為「定向選擇(guided selection)」之技術產生。在該方法中，使用所選的非人類單株抗體，例如小鼠抗體，引導能識別相同表位之完全人類抗體的選擇。A fully human antibody that recognizes a selected epitope can be produced using a technique known as "guided selection." In this method, selected non-human monoclonal antibodies, such as mouse antibodies, are used to direct selection of fully human antibodies that recognize the same epitope.

選擇性與主要係由序列識別號：5組成之胜肽結合的抗體可用於診斷，檢測受檢者體內登革熱病毒之包膜蛋白之存在，因此可檢測登革熱病毒之存在，而作為臨床測試步驟之一部分。所以，在一具體實施例中，本發明亦關於檢測受檢者體內登革熱病毒感染的方法，其包括：(a)從受檢者得到生物檢體；(b)使生物檢體與主要係由序列識別號：5組成之胜肽選擇性結合的抗體接觸；以及(c)檢測生物檢體中與該抗體單一性結合之抗原的存在。在一較佳具體實施例中，生物檢體為血液檢體，更佳為血清檢體，其係經由任何習知的方式從受檢者得到。The antibody which selectively binds to the peptide consisting of the sequence identification number: 5 can be used for diagnosis to detect the presence of the envelope protein of the dengue virus in the subject, thereby detecting the presence of the dengue virus as a clinical test procedure. portion. Therefore, in a specific embodiment, the present invention is also directed to a method of detecting a dengue virus infection in a subject, comprising: (a) obtaining a biological sample from the subject; and (b) causing the biological sample and the primary system to be SEQ ID NO: 5 antibody-selectively bound antibody contacts; and (c) detecting the presence of an antigen in a biological sample that binds singly to the antibody. In a preferred embodiment, the biological sample is a blood sample, more preferably a serum sample, which is obtained from the subject by any conventional means.

抗原存在之檢測可藉由各種免疫分析，諸如沉澱、被動凝集、酶-鍵結免疫吸附抗體(ELISA)技術及放射免疫分析(RIA)技術而進行。該檢測分析可藉由將抗體與可檢測之物質偶合而加速。可檢測物質之例子包括各種酶、彌補基團、螢光物質、發光物質、生物發光物質及放射活性物質。適當酶之例子包括辣根過氧化物酶、鹼性磷酸酶、β-半乳糖苷酶或乙醯膽鹼酯酶。適當彌補基團複合物之例子包括鏈黴抗生物素/生物素(streptavidin/biotin)以及抗生物素/生物素。適當的螢光物質之例子包括繖形酮(umbelliferone)、螢光素、螢光素異硫氰酸酯、羅丹明(rhodamine)、二氯三胺螢光素、丹磺醯氯(dansyl chloride)或藻紅蛋白(phycoerythrin)。發光物質之例子包括魯米諾(luminol)。生物發光物質之例子包括螢光素酶(luciferase)、冷光素(luciferin)及發光蛋白質(aequorin)。適當的放射活性物質之例子包括125 I、131 I、35 S或3 H。Detection of the presence of antigen can be performed by various immunoassays, such as precipitation, passive agglutination, enzyme-binding immunosorbent antibody (ELISA) techniques, and radioimmunoassay (RIA) techniques. This assay can be accelerated by coupling the antibody to a detectable substance. Examples of the detectable substance include various enzymes, compensating groups, fluorescent substances, luminescent substances, bioluminescent substances, and radioactive substances. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase or acetylcholinesterase. Examples of suitable compound complexes include streptavidin/biotin and avidin/biotin. Examples of suitable fluorescent materials include umbelliferone, luciferin, luciferin isothiocyanate, rhodamine, dichlorotriamine luciferin, dansyl chloride Or phycoerythrin. Examples of luminescent materials include luminol. Examples of bioluminescent substances include luciferase, luciferin, and aequorin. Examples of suitable radioactive materials include 125 I, 131 I, 35 S or 3 H.

在另一具體實施例中，本發明係關於在受檢者中檢測登革熱病毒之套組。該套組包括與主要係由序列識別號：5組成之胜肽選擇性結合的抗體以及說明在受檢者中使用該抗體檢測登革熱感染之說明書。該套組可為含有該組成物之容器，諸如瓶子、廣口瓶或盒子，連同附於容器上或含於容器內(諸如固定或印刷於容器之表面或標籤上)之說明書。該套組可進一步包括含有主要係由序列識別號：5組成之胜肽的製劑，以作為陽性對照組。In another embodiment, the invention relates to a kit for detecting dengue virus in a subject. The kit includes an antibody that selectively binds to a peptide consisting essentially of SEQ ID NO: 5 and instructions for detecting dengue infection using the antibody in a subject. The kit may be a container containing the composition, such as a bottle, a jar or a box, along with instructions attached to or contained within the container, such as on a surface or label that is fixed or printed on the container. The kit may further comprise a formulation comprising a peptide consisting essentially of SEQ ID NO: 5 as a positive control.

在受檢者中檢測登革熱病毒之另一方法涉及在受檢者之生物檢體中檢測與根據本發明具體實施例之抗原單一性結合之抗體的存在。該診斷方法包括：(a)從該受檢者得到生物檢體；(b)使該生物檢體與主要係由序列識別號：5組成之胜肽接觸；以及(c)檢測在該生物檢體中與該胜肽單一性結合之抗體的存在。在一較佳具體實施例中，該生物檢體為血液檢 體，較佳為血清檢體，其係經由任何習知的方式從受檢者得到。Another method of detecting dengue virus in a subject involves detecting the presence of an antibody that binds univocally to an antigen according to a particular embodiment of the invention in a subject's biopsy. The diagnostic method comprises: (a) obtaining a biological sample from the subject; (b) contacting the biological sample with a peptide consisting of a major sequence number: 5; and (c) detecting the biological test The presence of an antibody that binds to the peptide alone. In a preferred embodiment, the biological specimen is a blood test The body, preferably a serum sample, is obtained from the subject by any conventional means.

上述各種免疫分析可用於檢測分析。同樣地，該主要係由序列識別號：5組成之胜肽可與可檢測之物質偶合以利於檢測。The various immunoassays described above can be used for assays. Similarly, the peptide consisting essentially of sequence identifier: 5 can be coupled to a detectable substance to facilitate detection.

在另一具體實施例中，本發明係關於在受檢者中檢測登革熱病毒之套組，其包括主要係由序列識別號：5組成之胜肽以及說明在受檢者中使用該胜肽檢測登革熱感染之說明書。該套組可為含有該組成物之容器，諸如瓶子、廣口瓶或盒子，連同附於容器上或含於容器內(諸如固定或印刷於容器之表面或標籤上)之說明書。該套組可進一步包括含有選擇性地與主要係由序列識別號：5組成之胜肽結合的抗體的製劑，以作為陽性對照組。In another embodiment, the invention relates to a kit for detecting dengue virus in a subject, comprising a peptide consisting essentially of SEQ ID NO: 5 and illustrating the use of the peptide in a subject for detection Instructions for dengue infection. The kit may be a container containing the composition, such as a bottle, a jar or a box, along with instructions attached to or contained within the container, such as on a surface or label that is fixed or printed on the container. The kit may further comprise a formulation comprising an antibody that selectively binds to a peptide consisting essentially of SEQ ID NO: 5 as a positive control.

再者，根據本發明之具體實施例之抗體(或其片段)可用於治療登革熱感染。先前研究顯示E3結合之單株抗體為病毒吸附至Vero細胞之最有效的阻斷劑。主要係由序列識別號：5組成之胜肽為從四種血清型登革熱病毒而來之E3的共有序列。與胜肽單一性結合之抗體能與從四種血清型登革熱病毒而來之E3單一性結合。所以，該抗體可用於中和及阻斷由多種血清型登革熱病毒引起之感染。Furthermore, antibodies (or fragments thereof) according to particular embodiments of the invention can be used to treat dengue infection. Previous studies have shown that E3-bound monoclonal antibodies are the most potent blockers for viral adsorption to Vero cells. The peptide consisting mainly of SEQ ID NO: 5 is the consensus sequence of E3 from four serotypes of dengue virus. The antibody that binds to the peptide alone can bind to the E3 singularity from the four serotypes of dengue virus. Therefore, the antibody can be used to neutralize and block infections caused by various serotypes of dengue viruses.

本發明之一具體實施例係關於一種治療組成物，其包括與主要係由序列識別號：5組成之胜肽選擇性結合的抗體以及醫藥上可接受的載劑。於參照本揭示內容下，可將根據本發明之具體實施例之治療組成物依照製備醫藥上有用組成物的已知方法來調配。無菌磷酸鹽-緩衝食鹽水為醫藥上可接受之載劑之一例。其他可接受的載劑為本領域所熟知且可於參照本揭示內容下使用。One embodiment of the invention pertains to a therapeutic composition comprising an antibody that selectively binds to a peptide consisting essentially of SEQ ID NO: 5 and a pharmaceutically acceptable carrier. With reference to the present disclosure, therapeutic compositions in accordance with specific embodiments of the present invention can be formulated in accordance with known methods for preparing pharmaceutically useful compositions. Sterile phosphate-buffered saline is one example of a pharmaceutically acceptable carrier. Other acceptable carriers are well known in the art and can be used with reference to the present disclosure.

根據本發明具體實施例之抗體的調配物可以單位劑型存在，例如在多劑量容器中，且加入保存劑。組成物可採取在油性或水性載劑中之懸浮液、溶液或乳液等形式，且含有調配助劑諸如助懸浮劑、安定劑及/或分散劑。或者，活性成分可為粉末形式而於使用前用適當載劑，例如不含熱原的無菌水，加以重新配製。Formulations of antibodies according to particular embodiments of the invention may be presented in unit dosage form, such as in a multi-dose container, with the addition of a preservative. The composition may take the form of a suspension, solution or emulsion in an oily or aqueous vehicle, and may contain formulation aids such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient may be in powder form and reconstituted with a suitable carrier, such as sterile water without pyrogen, before use.

本發明之另一具體實施例係關於在受治者中治療登革熱疾病或異常之方法，其包括將治療有效量之根據本發明之具體實施例的治療組成物投予至受治者。在一具體實施例中，根據本發明之具體實施例之治療組成物可藉由靜脈內投予，例如經由濃注或連續輸注投予至受治者。在一些具體實施例中，將根據本發明之具體實施例之抗體單獨投予，或與至少一種或多種其他治療劑一起投予。該一起投予涵蓋在投予根據本發明之具體實施例之抗體之同時、之前或之後所投予的其他治療劑。在本領域中具有普通技術之人士於參照本揭示內容下，可輕易地決定投予特定治療劑之適當時機、順序及劑量。Another embodiment of the invention is directed to a method of treating a dengue disease or disorder in a subject comprising administering a therapeutically effective amount of a therapeutic composition according to a particular embodiment of the invention to a subject. In a specific embodiment, a therapeutic composition according to a particular embodiment of the invention can be administered to a subject by intravenous administration, for example, via bolus or continuous infusion. In some embodiments, antibodies according to particular embodiments of the invention are administered alone or with at least one or more other therapeutic agents. The combination is administered together with other therapeutic agents administered simultaneously, before or after administration of the antibodies according to particular embodiments of the invention. Appropriate timing, sequence, and dosage of a particular therapeutic agent can be readily determined by those of ordinary skill in the art with reference to the present disclosure.

本發明之再一具體實施例係關於一種設計對抗選自第1、2、3及4血清型組成之群中之至少一種登革熱病毒之胜肽疫苗之方法，該方法包括：(a)對於從選自第1、2、3及4血清型組成之群中之一種或多種登革熱病毒而來之E3之胺基酸序列，決定其共有序列；以及(b)設計包括具有該共有序列之胜肽之胜肽疫苗。A further embodiment of the invention relates to a method of designing a peptide vaccine against at least one dengue virus selected from the group consisting of serotypes 1, 2, 3 and 4, the method comprising: (a) An amino acid sequence derived from one or more of the serotypes of the first, second, third and fourth serotypes of E3, determining the consensus sequence thereof; and (b) designing a peptide comprising the consensus sequence The peptide vaccine.

參考下述之特定、非限定性實施例將可更了解本發明，但精於本技術人士當知此等實施例僅係例示說明本發明，而在隨附之申請專利範圍中將有更詳細的說明。The invention will be better understood by reference to the specific, non-limiting <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; instruction of.

實施例1Example 1 設計從四種血清型登革熱病毒而來之E蛋白質第三區塊的共有序列Designing a consensus sequence for the third block of E protein from four serotypes of dengue virus

首先對準來自該血清型之不同單離物序列，以決定從各血清型登革熱病毒而來之外套蛋白質第三區塊(E3)的共有序列。接著對準來自登革熱-1型、登革熱-2型、登革熱-3型及登革熱-4型病毒的共有序列，以決定來自所有四種血清型登革熱病毒之E3之共有序列，且用於設計對抗四種血清型登革熱病毒之胜肽疫苗。參照本發明揭示內容，可藉由本領域已知之各種方法取得E3之胺基酸序列，例如可從各種序列資料庫，或直接經由定序分析而取得。The different isolate sequences from this serotype are first aligned to determine the consensus sequence for the outer block of the protein (E3) from each serotype of dengue virus. The consensus sequence from dengue type-1, dengue type-2, dengue type-3 and dengue type 4 virus was then aligned to determine the consensus sequence of E3 from all four serotypes of dengue virus and was used to design against four A peptide vaccine for serotype dengue virus. With reference to the present disclosure, the amino acid sequence of E3 can be obtained by various methods known in the art, for example, from various sequence libraries, or directly via sequencing analysis.

如圖1所例示說明，E3之胺基酸序列首先經由National Center for Biotechnology Information (NCBI)之入口網站從GenBank® 得到。來自登革熱-1型病毒之E3之共有序列(DEN-1共有序列，序列識別號：1)藉由將來自DEN-1型病毒之不同單離物之6種胺基酸序列對準而得到(第1A圖)。該6種DEN-1胺基酸序列分別具有GenBank® 登錄號碼P17763、P27910、P27909、P33478、P27912及P27913。來自登革熱-2型病毒之E3之共有序列(DEN-2共有序列，序列識別號：2)藉由將來自DEN-2病毒之不同單離物之7種胺基酸序列對準而得到(第1B圖)。該7種DEN-2胺基酸序列分別具有GenBank® 登錄號碼P07564、P12823、Q9WDA6、P14337、P29990、P29991及P14340。來自登革熱-3型病毒之E3之共有序列(DEN-3共有序列，序列識別號：3)藉由將來自DEN-3病毒之不同單離物之5種胺基酸序列對準而得到(第1C圖)。該5種DEN-3胺基酸序列分別具有GeNBank® 登錄號碼P27915、Q99D35、Q5UB51、Q6YMS3及Q6YMS4。來自登革熱-4型病毒之E3之共有序列(DEN-4共有序列，序列識別號：4)藉由將來自DEN-4病毒之不同單離物之5種胺基酸序列對準而得到(第1D圖)。該5種DEN-4胺基酸序列分別具 有GenBank® 登錄號碼P09866、Q5UCB8、Q2YHF0、Q2YHF2及Q58HT7。如第1E圖所示，來自登革熱-1型、登革熱-2型、登革熱-3型及登革熱-4型病毒之E3之共有序列(DEN共有序列，序列識別號：5)藉由將來自四種血清型之各個之共有序列：即DEN-1共有序列(序列識別號：1)、DEN-2共有序列(序列識別號：2)、DEN-3共有序例(序列識別號：3)及DEN4-共有序列(序列識別號：4)對準而決定。As illustrated in Figure 1 case illustrated, E3 of the first amino acid sequence obtained from the GenBank ® via the National Center for Biotechnology Information (NCBI) of the portal. The consensus sequence of E3 from dengue type-1 virus (DEN-1 consensus sequence, SEQ ID NO: 1) was obtained by aligning the six amino acid sequences from different isolates of the DEN-1 type virus ( Figure 1A). The six DEN-1 amino acid sequences have GenBank ® accession numbers P17763, P27910, P27909, P33478, P27912, and P27913, respectively. The consensus sequence of E3 from dengue type 2 virus (DEN-2 consensus sequence, SEQ ID NO: 2) was obtained by aligning the seven amino acid sequences from different isolates of DEN-2 virus (p. 1B picture). The seven DEN-2 amino acid sequences have GenBank ® accession numbers P07564, P12823, Q9WDA6, P14337, P29990, P29991 and P14340, respectively. The consensus sequence of E3 from dengue type-3 virus (DEN-3 consensus sequence, SEQ ID NO: 3) was obtained by aligning the five amino acid sequences from different isolates of DEN-3 virus (p. 1C picture). The five kinds of DEN-3 amino acid sequences having GenBank ® registration number P27915, Q99D35, Q5UB51, Q6YMS3 and Q6YMS4. The consensus sequence of E3 from dengue type-4 virus (DEN-4 consensus sequence, SEQ ID NO: 4) was obtained by aligning the five amino acid sequences from different isolates of DEN-4 virus (p. 1D map). The five DEN-4 amino acid sequences have GenBank ® accession numbers P09866, Q5UCB8, Q2YHF0, Q2YHF2 and Q58HT7, respectively. As shown in Figure 1E, the consensus sequence (DEN consensus sequence, sequence number: 5) from E3 of dengue type-1, dengue type-2, dengue type-3, and dengue type 4 virus will come from four Consensus sequences for each serotype: DEN-1 consensus sequence (SEQ ID NO: 1), DEN-2 consensus sequence (SEQ ID NO: 2), DEN-3 consensus sequence (sequence ID: 3), and DEN4 - The consensus sequence (sequence identification number: 4) is determined by alignment.

精於本領域人士易於了解來自登革熱-1型、登革熱-2型、登革熱-3型及登革熱-4型病毒之ED III之共有序列(DEN共有序列)可依據DEN-1共有序列(序列識別號：1)、DEN-2共有序列(序列識別號：2)、DEN-3共有序列(序列識別號：3)及DEN4-共有序列(序列識別號：4)之對準而產生。舉例言之，DEN共有序列(序列識別號：5)之位置15可為四種胺基酸：L-麩胺酸(E)、L-纈草胺酸(V)、L-離胺酸(K)及L-天冬胺酸(D)之任一者；序列識別號5之位置27可為二種胺基酸：L-白胺酸(L)及L-纈草胺酸(V)之任一者；序列識別號5之位置28可為二種胺基酸：L-異白胺酸(I)及L-纈草胺酸(V)之任一者；序列識別號5之位置35可為四種胺基酸：L-蘇胺酸(T)、L-天冬胺酸(D)、L-麩胺酸(E)及L-丙胺酸(A)之任一者；以及序列識別號5之位置46可為二種胺基酸：L-麩醯胺酸(Q)、L-甲硫胺酸(M)、L-麩胺酸(E)及L-精胺酸(R)之任一者。本發明之具體實施例包括多種共有序列。於參照本揭示內容下，亦可將下述實施例應用於其他共有序列。It is easy for those skilled in the art to understand the consensus sequence of ED III from dengue type-1, dengue type-2, dengue type-3 and dengue type 4 virus (DEN consensus sequence) based on DEN-1 consensus sequence (sequence identification number) :1), DEN-2 consensus sequence (sequence identification number: 2), DEN-3 consensus sequence (sequence identification number: 3) and DEN4-consensus sequence (sequence identification number: 4) are generated by alignment. For example, position 15 of the DEN consensus sequence (SEQ ID NO: 5) can be four amino acids: L-glutamic acid (E), L-valeric acid (V), L-lysine ( Any of K) and L-aspartic acid (D); position 27 of SEQ ID NO: 5 can be two amino acids: L-leucine (L) and L-valeric acid (V) Any one of the positions 28 of the sequence identification number 5 may be any of two amino acids: L-isoleucine (I) and L-humulic acid (V); the position of the sequence number 5 35 may be any of four amino acids: L-threonine (T), L-aspartic acid (D), L-glutamic acid (E), and L-alanine (A); Position 46 of SEQ ID NO: 5 can be two amino acids: L-glutamic acid (Q), L-methionine (M), L-glutamic acid (E), and L-arginine ( R) either. Particular embodiments of the invention include a variety of consensus sequences. The following embodiments can also be applied to other consensus sequences with reference to the present disclosure.

實施例2Example 2 重組DEN共有蛋白質之製備Preparation of recombinant DEN consensus protein

OEN共有序列之基因係使用交疊的引子經由組裝PCR而得到。使用模板來擴增E3基因。正向5'- ACATATGAAAGGCATGAGCTAT GCG-3'引子包括Nde I位點以及反向引子5'-ACTCGAGGCTGCTG CCTTTTTTA-3'包括Xho I位點。結果，被表現之蛋白質之C-終端具有六-組胺酸標籤。如圖2A所示，使用Nde I及Xho I位點將PCR產物選殖入表現載體pET-22b(+)(Novagen, Madison, WI)中，產生質體pDconE3。重組DEN共有序列在大腸桿菌BL21Codon (DE3)中被表現。在BL21Codon (DE3)中誘生重組DEN共有序列後，將2.4公升之細胞培養物旋轉(8000 x g，歷20分鐘)且將小丸粒再懸浮於100ml均質化緩衝液(20 mM Tris-Cl (pH 8.0)，500 mM NaCl，10%甘油及50 mM蔗糖)中。用法式高壓細胞破壞機(French Press)於27 Kpsi破壞細胞(Constant Systems, Daventry, UK)後，細胞溶裂物藉由離心(80,000 x g，歷60分鐘)而澄清化。將上清液加載於20 ml Ni-NTA樹脂(Qiagen, San Diego, CA, USA)上。將該管柱(2.2 cm i.d. x 5.3 cm)用均質化緩衝液沖洗，然後用含有40 mM咪唑之相同緩衝液進一步沖洗。然後將該重組DEN共有序列用含500 mM咪唑之均質化緩衝液溶析。將該溶析出之部分對著20 mM Tris-Cl (pH 8.0)及1 mM EDTA透析。將該檢體加載在5 ml Q樹脂上以及收集通過該樹脂者。參考圖2B及2C，使用SDS-PAGE及充疫吸漬分析各步驟中之溶出分。且經純化之E3蛋白質可以SDS-PAGE凝膠中之帶(band)或免疫吸漬物(immunoblot顯現。The gene sequence of the OEN consensus sequence was obtained by assembly PCR using overlapping primers. A template was used to amplify the E3 gene. Forward 5'- The ACATATGAAAGGCATGAGCTAT GCG-3' primer includes the Nde I site and the reverse primer 5'-ACTCGAGGCTGCTG CCTTTTTTA-3' includes the Xho I site. As a result, the C-terminus of the expressed protein has a hexa-histidine tag. As shown in Figure 2A, PCR products were cloned into the expression vector pET-22b(+) (Novagen, Madison, WI) using Nde I and Xho I sites to generate plastid pDconE3. The recombinant DEN consensus sequence was expressed in E. coli BL21 Codon (DE3). After inducing the recombinant DEN consensus sequence in BL21Codon (DE3), 2.4 liters of cell culture was spun (8000 xg for 20 minutes) and the pellet was resuspended in 100 ml of homogenization buffer (20 mM Tris-Cl (pH) 8.0), 500 mM NaCl, 10% glycerol and 50 mM sucrose). After disrupting the cells (Constant Systems, Daventry, UK) at 27 Kpsi using a French Press High Pressure Cell Destructor (French Press), the cell lysate was clarified by centrifugation (80,000 x g for 60 minutes). The supernatant was loaded onto 20 ml of Ni-NTA resin (Qiagen, San Diego, CA, USA). The column (2.2 cm i.d. x 5.3 cm) was rinsed with homogenization buffer and then further rinsed with the same buffer containing 40 mM imidazole. The recombinant DEN consensus sequence was then eluted with a homogenization buffer containing 500 mM imidazole. The precipitated fraction was dialyzed against 20 mM Tris-Cl (pH 8.0) and 1 mM EDTA. The specimen was loaded on 5 ml of Q resin and collected through the resin. Referring to Figures 2B and 2C, the dissolution fractions in each step were analyzed using SDS-PAGE and vaccination. The purified E3 protein can be visualized by bands or immunosorbents in an SDS-PAGE gel.

實施例3Example 3 接種包括共有胜肽之疫苗的效果Vaccination of vaccines including consensus peptides

於實施例2之步驟之後，進行小鼠之疫苗接種以及收集接種疫苗後或免疫後的血清，亦從相同的動物收集接種疫苗前或免疫前的血清以作為對照組。接種疫苗之效果係藉由測量對抗四種血清型登革熱病毒之中和抗體反應而從接種疫苗後的血清。After the procedure of Example 2, vaccination of the mice and collection of vaccinated or post-immunized sera were also performed, and sera before or before immunization were also collected from the same animals as a control group. The effect of vaccination was obtained from vaccinated sera by measuring the neutralizing antibody response against four serotypes of dengue virus.

將BHK細胞，即廣泛地用作病毒宿主細胞的細胞，以2x105 細胞/孔之量接種在24-孔盤中並於37℃溫育整夜至產生匯合的單層。將已與預-免疫或免疫後血清混合的登革熱病毒以0.5ml的最終體積接種於單層中之細胞。預混合前的病毒滴度為約50至約250病毒斑點形成單位(FFU)/孔。該預混合於4℃進行整夜。讓病毒吸附於37℃進行2小時，且每15分鐘搖動該盤一次。於吸附結束時加入一由0.5 x RPMI-1640培養基(Sigma-Aldrich)、2.5%胎牛血清(FBS)及0.5%甲基纖維素組成之覆蓋層。將經感染之單層於37℃溫育。感染72至120小時後，從小孔移除覆蓋培養基以及將BHK細胞用冷磷酸鹽緩衝食鹽水(PBS)洗滌。將此等細胞用3.7%甲醛/PBS固定15分鐘。用PBS沖洗後，將細胞用0.1% nonidet NP-40/PBS於室溫可通透化15分鐘，以及用3%牛血清白蛋白(BSA)/PBS封阻30分鐘。在各孔中加入單株抗-登革熱抗體，然後於室溫溫育50分鐘。該單株抗-登革熱抗體衍生自融合瘤：美國類型培養物收集所(ATCC)登錄號HB-114之培養上清液。將其與登革熱病毒之所有成員反應。用PBS沖洗後，經抗體標記之細胞係藉由將該細胞與結合於辣根過氧化物酶(HRP)之二次抗體一起溫育30分鐘而加以檢測。用PBS沖洗後，藉由3,3,5,5'-四甲基聯苯胺(TMB)而顯現病毒斑點形成單位(FFU)。計算FFU以及藉由FFU之還原而測定抗血清之中和效果。The BHK cells, i.e., are widely used as viral host cells, in an amount of 2x10 5 cells / well of the seeded plate and was incubated at 37 ℃ overnight to produce a confluent monolayer in 24-well. The dengue virus that has been mixed with the pre-immunized or post-immune serum is seeded in a single layer in a final volume of 0.5 ml. The virus titer prior to premixing is from about 50 to about 250 viral spot forming units (FFU) per well. The premix was carried out overnight at 4 °C. The virus was allowed to adsorb at 37 ° C for 2 hours, and the disk was shaken every 15 minutes. At the end of the adsorption, a cover layer consisting of 0.5 x RPMI-1640 medium (Sigma-Aldrich), 2.5% fetal bovine serum (FBS) and 0.5% methylcellulose was added. The infected monolayer was incubated at 37 °C. After 72 to 120 hours of infection, the overlay medium was removed from the wells and BHK cells were washed with cold phosphate buffered saline (PBS). The cells were fixed with 3.7% formaldehyde/PBS for 15 minutes. After washing with PBS, cells were permeabilized with 0.1% nonidet NP-40/PBS for 15 minutes at room temperature and blocked with 3% bovine serum albumin (BSA)/PBS for 30 minutes. Individual anti-dengue antibodies were added to each well and incubated for 50 minutes at room temperature. The monoclonal anti-dengue antibody was derived from a culture supernatant of a fusion tumor: American Type Culture Collection (ATCC) Accession No. HB-114. It is reacted with all members of the dengue virus. After washing with PBS, the antibody-labeled cells were detected by incubating the cells with a secondary antibody conjugated to horseradish peroxidase (HRP) for 30 minutes. After washing with PBS, virus spot forming units (FFU) were visualized by 3,3,5,5'-tetramethylbenzidine (TMB). The FFU and the antisera neutralization effect were determined by reduction of the FFU.

如圖3所示，當將BHK細胞與已和各種血清稀釋度之免疫後血清預混合的登革熱-1型、登革熱-2型、登革熱-3型及登革熱-4型病毒溫育時，未檢測到FFU。在各治療組中進行2至3次複製。此等結果顯示序列識別號：5之共有胜肽在接種疫苗的動物中能誘生出對抗登革熱病毒之四種血清型的中和抗體反應。As shown in Figure 3, when BHK cells were incubated with dengue-1, dengue-2, dengue-3 and dengue-4 viruses that had been pre-mixed with various serum dilutions of immunized serum, no FFU was detected. . Two to three replicates were performed in each treatment group. These results show that the consensus peptide of sequence number: 5 can induce a neutralizing antibody response against the four serotypes of dengue virus in vaccinated animals.

實施例4Example 4 由衍生自DEN共有序列之胜肽所誘生的中和抗體反應Neutralizing antibody response induced by a peptide derived from the DEN consensus sequence

合成胜肽之序列從具序列識別號5之DEN共有序列之位置7至40衍生。起初先用30μg之乳化在完全弗氏佐劑中之合成DEN共有胜肽(序列識別號：6)經由皮下將小鼠免疫。於初始免疫後第14及28日，使用相同量之乳化在不完全弗氏佐劑中之合成胜肽追加免疫。第二次追加後2星期從小鼠收集含有對抗DEN共有胜肽(序列識別號：6)之抗體的血清。製備接種疫苗前之血清(naive sera)以作為對照組。藉由對抗登革熱病毒之四種血清型的中和抗體反應從接種疫苗後之血清測量接種疫苗之效果。The sequence of the synthetic peptide is derived from positions 7 to 40 of the DEN consensus sequence having SEQ ID NO: 5. Initially, mice were immunized subcutaneously with 30 μg of the synthetic DEN consensus peptide (SEQ ID NO: 6) emulsified in complete Freund's adjuvant. On the 14th and 28th day after the initial immunization, the same amount of emulsification was used for additional immunization with the synthetic peptide in incomplete Freund's adjuvant. The serum containing the antibody against the DEN consensus peptide (SEQ ID NO: 6) was collected from the mice 2 weeks after the second addition. Serum (naive sera) before vaccination was prepared as a control group. The effect of the vaccination was measured from the vaccinated serum by a neutralizing antibody response against the four serotypes of the dengue virus.

如圖4所示，當將BHK細胞分別與已和各種血清稀釋度之免疫後血清預混合的登革熱-1型、登革熱-2型、登革熱-3型及登革熱-4型病毒溫育時，檢測到之FFU之數目顯著減少。在各治療組中進行2至3次複製。此等結果顯示序列識別號：6之合成胜肽在接種疫苗的動物中能誘生出對抗登革熱病毒之四種血清型的中和抗體反應。As shown in Fig. 4, when BHK cells were separately incubated with dengue type-1, dengue type-2, dengue type-3 and dengue type 4 viruses which were premixed with various serum dilutions of immunized serum, they were detected. The number of FFUs is significantly reduced. Two to three replicates were performed in each treatment group. These results show that the synthetic peptide of sequence number: 6 can induce a neutralizing antibody response against the four serotypes of dengue virus in vaccinated animals.

精於本領域之人士當了解可在不偏離本發明之廣義概念下對於上述具體實施例進行改變。所以，應了解本發明不限於所揭示之特定具體實施例，但其意欲涵蓋在如隨附之申請專利範圍所界定之本發明之精神及範圍內的修改。It will be apparent to those skilled in the art that changes may be made to the specific embodiments described above without departing from the broad scope of the invention. Therefore, it is understood that the invention is not limited to the specific embodiments disclosed, but is intended to cover the modifications of the scope of the invention as defined by the appended claims.

上述摘要以及下述本發明之較佳具體實施例之詳細說明，當與所附圖式一起閱讀時，將更易被了解。就說明本發明之目的而言，目前較佳的具體實施例呈現於圖式中。不過，應了解本發明不限於所示之確切具體實施例。The above summary, as well as the following detailed description of the preferred embodiments of the invention, For the purposes of the present invention, the presently preferred embodiments are presented in the drawings. However, it is to be understood that the invention is not to be limited

在圖式中：圖1係說明根據本發明具體實施例用於對抗四種血清型登革熱病毒之胜肽疫苗；圖2A顯示根據本發明具體實施例之質體pDconE3之示意構築體；圖2B為顯示根據本發明具體實施例之純化E3蛋白質之SDS-PAGE凝膠的照片；圖2C為顯示根據本發明具體實施例之純化E3蛋白質之免疫吸漬圖；圖3為顯示當BHK細胞以登革熱-1型、登革熱-2型、登革熱-3型及登革熱-4型病毒感染時所檢測到之病毒斑點形成單位(FFU)之長條圖，其中該等病毒已與從用包括序列識別號：5之DEN共有序列之疫苗所接種之動物而來的疫苗接種後血清之各種稀釋液預先混合，或已與根據本發明具體實施例之接種疫苗前之血清之各種稀釋液預先混合；以及圖4為顯示當BHK細胞以登革熱-1型、登革熱-2型、登革熱-3型及登革熱-4型病毒感染時所檢測到之FFU之長條圖，其中該等病毒已與從用序列識別號：6之合成胜肽所接種之動物而來的疫苗接種後血清之各種稀釋液預先混合，或已與根據本發明具體實施例之接種疫苗前血清之各種稀釋液預先混合。In the drawings: Figure 1 illustrates a peptide vaccine for combating four serotypes of dengue virus in accordance with an embodiment of the present invention; Figure 2A shows a schematic construct of a plastid pDconE3 in accordance with an embodiment of the present invention; A photograph showing an SDS-PAGE gel of purified E3 protein according to a specific embodiment of the present invention; FIG. 2C is a diagram showing immunoabsorption of purified E3 protein according to a specific embodiment of the present invention; and FIG. 3 is a diagram showing when BHK cells are dengue- A bar graph of the virus spot forming unit (FFU) detected by type 1 infection, dengue type-2, dengue type-3 and dengue type 4 virus infection, wherein the viruses have been used with the sequence identification number: 5 The various dilutions of the vaccinated serum from the vaccinated animal of the DEN consensus sequence are pre-mixed or pre-mixed with various dilutions of the pre-vaccination serum according to a specific embodiment of the present invention; A bar graph showing the FFU detected when BHK cells are infected with dengue type-1, dengue type-2, dengue type-3, and dengue type 4 virus, wherein the viruses have been used with the sequence identification number: 6 The various dilutions of sera previously mixed after vaccination vaccinated animals from the synthetic peptides, or premixed with a diluent in accordance with various embodiments of the sera before vaccination vaccine particular embodiment of the present invention.

<110> Chen,Hsin-Wei Chong,Choi-Sing Leng,Chih-Hsiang<110> Chen, Hsin-Wei Chong, Choi-Sing Leng, Chih-Hsiang

<120> 登革熱病毒胜肽疫苗及其製備與使用方法<120> Dengue virus peptide vaccine and preparation and use method thereof

<130> 6819380027US<130> 6819380027US

<150> US 60/942,703<150> US 60/942,703

<151> 2007-06-08<151> 2007-06-08

<160> 6<160> 6

<170> PatentIn version 3.3<170> PatentIn version 3.3

<210> 1<210> 1

<211> 103<211> 103

<212> PRT<212> PRT

<213> 登革熱病毒-1型<213> Dengue virus type-1

<400> 1 <400> 1

<210> 2<210> 2

<211> 103<211> 103

<212> PRT<212> PRT

<213> 登革熱病毒-2型<213> Dengue Virus-2

<400> 2 <400> 2

<210> 3<210> 3

<211> 103<211> 103

<212> PRT<212> PRT

<213> 登革熱病毒-3型<213> Dengue Virus-3

<400> 3 <400> 3

<210> 4<210> 4

<211> 103<211> 103

<212> PRT<212> PRT

<213> 登革熱病毒-4型<213> Dengue Virus Type-4

<400> 4 <400> 4

<210> 5<210> 5

<211> 103<211> 103

<212> PRT<212> PRT

<213> 人造<213> Artificial

<220><220>

<223> 登革熱病毒之共有序列<223> Consensus sequence of dengue virus

<220><220>

<221> MISC_FEATURE<221> MISC_FEATURE

<222> (15)..(15)<222> (15)..(15)

<223> Xaa可為Glu,Val,Lys,Asp<223> Xaa can be Glu, Val, Lys, Asp

<220><220>

<221> MISC_FEATURE<221> MISC_FEATURE

<222> (27)..(27)<222> (27)..(27)

<223> Xaa可為Leu,Val<223> Xaa can be Leu, Val

<220><220>

<221> MISC_FEATURE<221> MISC_FEATURE

<222> (28)..(28)<222> (28)..(28)

<223> Xaa可為Isoleu,Val<223> Xaa can be Isoleu, Val

<220><220>

<221> MISC_FEATURE<221> MISC_FEATURE

<222> (35)..(35)<222> (35)..(35)

<223> Xaa可為Thr,Asp,Glu,Ala<223> Xaa can be Thr, Asp, Glu, Ala

<220><220>

<221> MISC_FEATURE<221> MISC_FEATURE

<222> (46)..(46)<222> (46)..(46)

<223> Xaa可為Gln,Met,Glu,Arg<223> Xaa can be Gln, Met, Glu, Arg

<400> 5 <400> 5

<210> 6<210> 6

<211> 34<211> 34

<212> PRT<212> PRT

<213> 人造<213> Artificial

<220><220>

<223> 合成胜肽<223> Synthetic peptide

<400> 6 <400> 6

Claims (24)

一種經單離之胜肽，其為序列識別號：5。 An isolated peptide which is a sequence identifier: 5. 一種重組胜肽，其包括序列識別號：5。 A recombinant peptide comprising a sequence identifier: 5. 一種合成胜肽，其為序列識別號：5。 A synthetic peptide which is a sequence identifier: 5. 如申請專利範圍第3項所述之合成胜肽，其為序列識別號：6。 The synthetic peptide described in claim 3, which is the sequence identifier: 6. 一種免疫組成物，其包括序列識別號：5之胜肽。 An immunological composition comprising a peptide of sequence number: 5. 如申請專利範圍第5項所述之免疫組成物，其中該胜肽主要係由序列識別號：6組成。 The immunological composition of claim 5, wherein the peptide is mainly composed of a sequence identifier: 6. 如申請專利範圍第5項所述之免疫組成物，其中每一投予劑量包括約0.5微克至約1毫克之該胜肽。 The immunological composition of claim 5, wherein each dose comprises from about 0.5 microgram to about 1 milligram of the peptide. 如申請專利範圍第5項所述之免疫組成物，其中更包括佐劑。 The immunological composition of claim 5, which further comprises an adjuvant. 如申請專利範圍第5項所述之免疫組成物，其中該免疫組成物為對抗選自第1、2、3及4血清型所組成之群中之至少一種登革熱病毒之疫苗。 The immunological composition according to claim 5, wherein the immunological composition is a vaccine against at least one dengue virus selected from the group consisting of the serotypes 1, 2, 3 and 4. 如申請專利範圍第9項所述之免疫組成物，其中該疫苗為對抗第1、2、3及4血清型中之所有四種登革熱病毒。 The immunological composition of claim 9, wherein the vaccine is against all four dengue viruses of the first, second, third and fourth serotypes. 一種套組，其包括申請專利範圍第5項之免疫組成物以及說明使用該免疫組成物以誘發受治者免疫反應之說明書。 A kit comprising the immunological composition of claim 5 and instructions for using the immunological composition to induce an immune response in the subject. 如申請專利範圍第9項所述之免疫組成物，其中更包括至少另一種疫苗，係對抗至少另一種感染物或係藉由另一作用機制誘發對抗至少一種血清型登革熱病毒之保護性反應。 The immunological composition of claim 9 further comprising at least one other vaccine against a at least one other infectious agent or a protective response against at least one serotype of dengue virus by another mechanism of action. 一種經實質純化之抗體，其選擇性與序列識別號：5之胜肽結合。 A substantially purified antibody having a selectivity that binds to a peptide of sequence number: 5. 一種製造選擇性與序列識別號：5之胜肽結合之抗體的方法，該方法包括： (a)使細胞與該胜肽接觸；以及(b)於允許該細胞產生抗體之條件下培養該細胞。 A method of making an antibody that selectively binds to a peptide of sequence number: 5, the method comprising: (a) contacting the cell with the peptide; and (b) culturing the cell under conditions that allow the cell to produce an antibody. 如申請專利範圍第14項所述之方法，其中該細胞係存在於動物體內。 The method of claim 14, wherein the cell line is present in the animal. 一種醫藥組成物，其包括與序列識別號：5之胜肽選擇性結合的抗體。 A pharmaceutical composition comprising an antibody that selectively binds to a peptide of sequence number: 5. 一種檢測受檢者之登革熱病毒感染之方法，該方法包括：(a)從該受檢者取得生物檢體；(b)使該生物檢體與主要係由序列識別號：5組成之胜肽選擇性結合的抗體接觸；以及(c)檢測在該生物檢體中與該抗體專一性結合之抗原的存在。 A method for detecting a dengue virus infection of a subject, the method comprising: (a) obtaining a biological sample from the subject; (b) causing the biological sample to be a peptide consisting of a sequence identifier of: Selectively bound antibody contact; and (c) detecting the presence of an antigen that specifically binds to the antibody in the biological sample. 如申請專利範圍第17項所述之方法，其中該登革熱病毒為選自第1、2、3及4血清型組成之群中之至少一種。 The method of claim 17, wherein the dengue virus is at least one selected from the group consisting of serotypes 1, 2, 3 and 4. 如申請專利範圍第17項所述之方法，其中該受檢者為人類受檢者。 The method of claim 17, wherein the subject is a human subject. 一種檢測受檢者之登革熱病毒感染之方法，該方法包括：(a)從該受檢者取得生物檢體；(b)使該生物檢體與主要係由序列識別號：5組成之胜肽接觸；以及(c)檢測在該生物檢體中與該胜肽專一性結合之抗體的存在。 A method for detecting a dengue virus infection of a subject, the method comprising: (a) obtaining a biological sample from the subject; (b) causing the biological sample to be a peptide consisting of a sequence identifier of: Contacting; and (c) detecting the presence of an antibody that specifically binds to the peptide in the biological sample. 如申請專利範圍第20項所述之方法，其中該登革熱病毒為選自第1、2、3及4血清型組成之群中至少一種。 The method of claim 20, wherein the dengue virus is at least one selected from the group consisting of serotypes 1, 2, 3, and 4. 如申請專利範圍第20項所述之方法，其中該受檢者為人類受檢者。 The method of claim 20, wherein the subject is a human subject. 一種檢測受檢者之登革熱病毒感染之套組，其包括：主要係由序列識別號：5組成之胜肽以及說明使用該胜肽檢測該受檢者體內登革熱病毒感染之說明書。 A kit for detecting a dengue virus infection of a subject, comprising: a peptide mainly consisting of SEQ ID NO: 5 and instructions for detecting dengue virus infection in the subject using the peptide. 一種檢測受檢者之登革熱病毒感染之套組，其包括：選擇性與主要係由序列識別號：5組成之胜肽結合的抗體以及說明使用該抗體檢測該受檢者體內登革熱病毒感染之說明書。A kit for detecting a dengue virus infection of a subject, comprising: an antibody selectively binding to a peptide consisting mainly of SEQ ID NO: 5, and instructions for detecting dengue virus infection in the subject using the antibody .
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